NON-PCR BASED MOLECULAR

APPROACHES FOR DETECTION AND

IDENTIFICATION OF PLANT PATHOGENSDOCTORAL SEMINAR II

PUJA PANDEY41124

1

INTRODUCTION

Plant Pathology

Molecular Plant Pathology

2

bull Visual observations

bull Cultural

bull Microscopy

CONVENTIONAL TECHNIQUES

3

VISUAL OBSERVATION

SIGNS AND SYMPTOMS

Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans

Citrus canker

4

Problem with biotic and abiotic factors

5

CULTURAL LABORATORY TECHNIQUES

Bacterial ooze

Cultures of Ralstonia solanacearum

6

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

INTRODUCTION

Plant Pathology

Molecular Plant Pathology

2

bull Visual observations

bull Cultural

bull Microscopy

CONVENTIONAL TECHNIQUES

3

VISUAL OBSERVATION

SIGNS AND SYMPTOMS

Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans

Citrus canker

4

Problem with biotic and abiotic factors

5

CULTURAL LABORATORY TECHNIQUES

Bacterial ooze

Cultures of Ralstonia solanacearum

6

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

bull Visual observations

bull Cultural

bull Microscopy

CONVENTIONAL TECHNIQUES

3

VISUAL OBSERVATION

SIGNS AND SYMPTOMS

Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans

Citrus canker

4

Problem with biotic and abiotic factors

5

CULTURAL LABORATORY TECHNIQUES

Bacterial ooze

Cultures of Ralstonia solanacearum

6

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

VISUAL OBSERVATION

SIGNS AND SYMPTOMS

Rot - Sclerotium rolfsiiLate blight of potato ndash Phytophthora infestans

Citrus canker

4

Problem with biotic and abiotic factors

5

CULTURAL LABORATORY TECHNIQUES

Bacterial ooze

Cultures of Ralstonia solanacearum

6

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Problem with biotic and abiotic factors

5

CULTURAL LABORATORY TECHNIQUES

Bacterial ooze

Cultures of Ralstonia solanacearum

6

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

CULTURAL LABORATORY TECHNIQUES

Bacterial ooze

Cultures of Ralstonia solanacearum

6

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Fungal cultureshellip

Alternaria culture Fusarium culture

7

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

MICROSCOPY An Aid To

The Eyes

Microscope

Stereoscopic Microscope

Fluorescence Microscope

Light Microscope

Bright Field Dark FieldPhase

ContrastConfocal

Electron Microscope

Scanning electron

Microscope

Transmission electron

Microscope

8

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Types of Microscope

1) Stereoscopic microscope

Function Visible light to illuminate the surface

of a sample (2000x ) without disrupting them

2) Compound microscope (light)

Function Visible light to illuminate a thin

section of sample cells and tissues

3) Confocal laser scanning fluorescence

microscope

Function Thin lsquoslicesrsquo in a sample while

keeping sample intact

Specifically at parts of a cell (such as

individual proteins) by labelling them with

fluorescence

9

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Scanning Electron Microscope

Function Surface of objects at high resolution

(3D image - 500 000x )

Principle Beam of electrons being knocked off

the surface of the sample and then picked up by a

detector

Why we use SEM

The SEM probably gives the best depth of field

out of any microscope

10

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Transmission Electron Microscope

Function very thin cross-section of an

object (cell) internal structure of objects

high resolution (500 000x )

Principle Electrons pass through the

sample and some are deflected and some

pass right through and that forms our

image which is focussed on objective

lens

11

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Microscopy Techniques Applied to the Study of Phytoplasma

Diseases Traditional and Innovative MethodsRita Musetti and Maria Augusta Favali

12

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Light Microscopy

Dienesrsquostain was first developed

as a specific stain for animal

mycoplasma colonies

Phloem tissues of stems infected

by phytoplasmas stained dark

blue while xylem was turquoise

and cortex light blue

Musetti and Favali 2004

13

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Fluorescence Microscopy

DAPI staining of hand cut

sections of healthy

Antibody plus fluorochrome

such as fluorescein

isothiocyanate (FITC) and

stained

Phytoplasma-infected

the fluorescent bright spots

visible at phloem level

Musetti and Favali 200414

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Transmission electron microscopy (TEM)

Phytoplasmas in the phloem

cells of Catharanthus roseus

Musetti and Favali 200415

Phytoplasmas in the phloem of

apple tissues

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Immuno-electron microscopy (IEM) of thin sections

Phytoplasmas in phloem tissues of Catharanthus roseus L embeded with primary

monoclonal antibody and gold Labelledsecondary antibody

Gold partcle (15 nm) few

particles are visible on

phytoplasma membrane

Gold partcle (5 nm) particles are

well distributed over the periphery

of the phytoplasmas

Musetti and Favali 200416

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

High resolution autoradiography

Phytoplasmas in phloem cells of white clover (Trifolium repens L) after 3

hours labelling with thymidine-3H The silver grains were seen on the

dividing phytoplasmas

17

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

Staining Technique For Histopathological Tests

bull Staining is an auxiliary technique used in microscopy to

enhance contrast in the microscopic image

eg Crystal violet stains only Gram positive bacteria

18

19

Histopathological analysis of infected tissues

20

Valencia sweet

oranges infected

with

Colletotrichum

acutatum

Valenciasweet orange

fruits infected

with Guignardia

citricarpa

Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013

21

Limitations Faced due to conventional techniques

Latent infection eg Potato ring rot

Misleading infection eg Black lesions (Alternaria) and bacterial

blight of carrot (Xanthomonas)

Co-infection Alteration of symptoms

SEROLOGICAL METHODS ndash De Vorac

Antigen Antibody

Antigen Antibody

Positive

Result 22

Antigen

A molecule usually a protein when it is injected into a

warm blooded animal produces antibody (immune

response)

Antibody

A molecule produced in a warm

blooded serum of animal in

response to the stimulus antigens

Antibodies are immune system-

related proteins called

immunoglobulin

23

Variable region composed of 110-

130 amino acids give the antibody

its specificity for binding antigen

Variable region includes the ends of

the light and heavy chains

Constant region determines the

mechanism used to destroy antigen

Structure of Antibody

24

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Histopathological analysis of infected tissues

20

Valencia sweet

oranges infected

with

Colletotrichum

acutatum

Valenciasweet orange

fruits infected

with Guignardia

citricarpa

Toluidine blue staining Toluidine blue plus safranin staining Marques et al 2013

21

Limitations Faced due to conventional techniques

Latent infection eg Potato ring rot

Misleading infection eg Black lesions (Alternaria) and bacterial

blight of carrot (Xanthomonas)

Co-infection Alteration of symptoms

SEROLOGICAL METHODS ndash De Vorac

Antigen Antibody

Antigen Antibody

Positive

Result 22

Antigen

A molecule usually a protein when it is injected into a

warm blooded animal produces antibody (immune

response)

Antibody

A molecule produced in a warm

blooded serum of animal in

response to the stimulus antigens

Antibodies are immune system-

related proteins called

immunoglobulin

23

Variable region composed of 110-

130 amino acids give the antibody

its specificity for binding antigen

Variable region includes the ends of

the light and heavy chains

Constant region determines the

mechanism used to destroy antigen

Structure of Antibody

24

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

21

Limitations Faced due to conventional techniques

Latent infection eg Potato ring rot

Misleading infection eg Black lesions (Alternaria) and bacterial

blight of carrot (Xanthomonas)

Co-infection Alteration of symptoms

SEROLOGICAL METHODS ndash De Vorac

Antigen Antibody

Antigen Antibody

Positive

Result 22

Antigen

A molecule usually a protein when it is injected into a

warm blooded animal produces antibody (immune

response)

Antibody

A molecule produced in a warm

blooded serum of animal in

response to the stimulus antigens

Antibodies are immune system-

related proteins called

immunoglobulin

23

Variable region composed of 110-

130 amino acids give the antibody

its specificity for binding antigen

Variable region includes the ends of

the light and heavy chains

Constant region determines the

mechanism used to destroy antigen

Structure of Antibody

24

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

SEROLOGICAL METHODS ndash De Vorac

Antigen Antibody

Antigen Antibody

Positive

Result 22

Antigen

A molecule usually a protein when it is injected into a

warm blooded animal produces antibody (immune

response)

Antibody

A molecule produced in a warm

blooded serum of animal in

response to the stimulus antigens

Antibodies are immune system-

related proteins called

immunoglobulin

23

Variable region composed of 110-

130 amino acids give the antibody

its specificity for binding antigen

Variable region includes the ends of

the light and heavy chains

Constant region determines the

mechanism used to destroy antigen

Structure of Antibody

24

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Antigen

A molecule usually a protein when it is injected into a

warm blooded animal produces antibody (immune

response)

Antibody

A molecule produced in a warm

blooded serum of animal in

response to the stimulus antigens

Antibodies are immune system-

related proteins called

immunoglobulin

23

Variable region composed of 110-

130 amino acids give the antibody

its specificity for binding antigen

Variable region includes the ends of

the light and heavy chains

Constant region determines the

mechanism used to destroy antigen

Structure of Antibody

24

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Variable region composed of 110-

130 amino acids give the antibody

its specificity for binding antigen

Variable region includes the ends of

the light and heavy chains

Constant region determines the

mechanism used to destroy antigen

Structure of Antibody

24

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

MONOCLONAL ANTIBODY

POLYCLONAL ANTIBODY

Composed of a variety of antibody

Have multiple epitopes

Antibody derived from a single

clone and specific for a single

epitope

Consist of single type of antibody

Produced by hybridoma technique

Small quantity of antigen is enough

for development

25

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Production of Mabs by Hybridoma Technique

26

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Antigen antibody based technique

Direct test

Precipitation test

1 Tube precipitation

2 Ring precipitation

Micro Precipitation test

Agglutination tests

1 Chloroplast agglutination

2 Latex agglutination

Gel diffusion test

Immuno-electrophoresis

Indirect test

ELISA test

1 Direct ELISA (DAS ELISA)

2 Indirect (DAC ELISA)

3 DIBA ELISA

4 Lateral flow device

Immunofluorescence

Immuno Sorbent Electron Microsopy(ISEM)

Flow cytometry

27

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Tube precipitation test

Widely used

Reactants diluted in 85gl

NaCl followed by

incubation at 37degC in water

bath

Observations

If elongated virus particle -

floccular

If spherical virus particle -

granular

Precipitation test

28

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Done on a micro-scale to

economize on antiserum

Drop of dilution mixture (antiserum

amp virus suspension) are mixed at

bottom of a Petri plate

The precipitates produced are

observed with a microscope with

dark-ground illumination

Precipitation varies depending on

the ratio of concentration of antigen

and antibody

Micro precipitition test

29

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Latex agglutination

30

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Chloroplast agglutination

Crude fresh leaf sap from diseased plant

Antiserum

Chloroplast fragments

clump together

31

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

The reactants antiserum and virus solution are placed in well cut

in the agar (containg 085 NaCl and 002 sodium azide) in

Petri plate

Antibody and virus diffuse into the agar from the adjacent wells

Where they meet precipitation zones in the form of white band

are formed

Gel diffusion test

Oservation

a) Bands Identical or closely related

b) Spurs Distantly Related

c) Intersect Unrelated 32

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Radial or single diffusion

Double or Ouchterlony diffusion33

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Immuno-electrophoresis

34

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

ELISA Enzyme- Linked Immuno-Sorbent Assay

ELISA was initially applied for plant viruses by MF Clark and

Adams (1976)

Sensitive detects at concentration of 1-10 ngml

It involves an enzyme-mediated colour change reaction to detect

antibody binding

Degree of colour change usually measured quantitatively in

spectrophotometer at 405 nm

Ward et al 200335

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

DAS ELISA Double antibody sandwich ELISA

Direct ELISA

First time describe by Clark and Adams in 1977

p-nitrophenyl

phosphatep-nitrophenol Ward et al 2003

36

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

DAC ELISA direct antigen coating ELISA

Indirect ELISA

Stand for Easy to rapid assay

Ward et al 200337

Combination of electron microscopy

and serology

First time described by Derrick in1973

Virus and antiserum are reacted

together

Antigen are trapped onto grid coated

with specific antiserum negatively

stained (Uranyl acetate -1) and the

result viewed in the EM

Immuno Sorbernt electron microscope (ISEM)

Tubular particle of beet

necrotic yellow vein virus

38

Dot Immunoblotting Assay (DIBA) OR Dot ELISA

Substrate Nitro tetrazolium

BCIP

39

Lateral flow technique

bull The principles used for rapid lateral flow devices are primarily

those of ELISA

bull Various types of filters are used as the solid support for the

initial binding reaction

A lateral flow device test kit developed by Central Science

Laboratory UK permits detection of R solanacearum in a 3-

minute40

41

Ouchterloniersquos double diffusion test

I ndash Healthy cane

extract

II ndash Control blood

serum of rabbit

III IV V ndash Antigen

of host pathogen

A ndash Antibody

raised against host

pathogen

Lingayya and Naik 2002

42

Detection of Colletotrichum falcatum infection in sugarcane

tissue by DAC - ELISA

Lingayya and Naik 2002 43

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Dot Immunoblotting Assay (DIBA) OR Dot ELISA

Substrate Nitro tetrazolium

BCIP

39

Lateral flow technique

bull The principles used for rapid lateral flow devices are primarily

those of ELISA

bull Various types of filters are used as the solid support for the

initial binding reaction

A lateral flow device test kit developed by Central Science

Laboratory UK permits detection of R solanacearum in a 3-

minute40

41

Ouchterloniersquos double diffusion test

I ndash Healthy cane

extract

II ndash Control blood

serum of rabbit

III IV V ndash Antigen

of host pathogen

A ndash Antibody

raised against host

pathogen

Lingayya and Naik 2002

42

Detection of Colletotrichum falcatum infection in sugarcane

tissue by DAC - ELISA

Lingayya and Naik 2002 43

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Lateral flow technique

bull The principles used for rapid lateral flow devices are primarily

those of ELISA

bull Various types of filters are used as the solid support for the

initial binding reaction

A lateral flow device test kit developed by Central Science

Laboratory UK permits detection of R solanacearum in a 3-

minute40

41

Ouchterloniersquos double diffusion test

I ndash Healthy cane

extract

II ndash Control blood

serum of rabbit

III IV V ndash Antigen

of host pathogen

A ndash Antibody

raised against host

pathogen

Lingayya and Naik 2002

42

Detection of Colletotrichum falcatum infection in sugarcane

tissue by DAC - ELISA

Lingayya and Naik 2002 43

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

41

Ouchterloniersquos double diffusion test

I ndash Healthy cane

extract

II ndash Control blood

serum of rabbit

III IV V ndash Antigen

of host pathogen

A ndash Antibody

raised against host

pathogen

Lingayya and Naik 2002

42

Detection of Colletotrichum falcatum infection in sugarcane

tissue by DAC - ELISA

Lingayya and Naik 2002 43

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Ouchterloniersquos double diffusion test

I ndash Healthy cane

extract

II ndash Control blood

serum of rabbit

III IV V ndash Antigen

of host pathogen

A ndash Antibody

raised against host

pathogen

Lingayya and Naik 2002

42

Detection of Colletotrichum falcatum infection in sugarcane

tissue by DAC - ELISA

Lingayya and Naik 2002 43

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Detection of Colletotrichum falcatum infection in sugarcane

tissue by DAC - ELISA

Lingayya and Naik 2002 43

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

TDA = 3 X standard deviation of

healthy sample + mean value for

healthy sample

44

FUNGUS

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Advantage of ELISA

It is sensitive

Semiautomatic technique

Application against large number of sample

Reproducible

Qualitative amp Quantitative

Suitable for automation high speed

No radiation hazards

45

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Immunofluorescence

The intercellular location amp

distribution of viruses

Globulins mixed with a

fluorescent dye (Fluorescein

isothiocyanate and Rhodamine

B)

Introduced into the infected

cellstissue with antigen and

antibody reaction fluorescence

takes place

46

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Flow cytometry

Cell suspensions are filtered to remove large particles then

stained with fluorochrome-labelled antibodies

Fluorescent markers for viability

Stains such as propidium and

hexidium iodide for red fluorescent

staining of dead cells

Carboxy fluorescein diacetate and calcein

AM for green fluorescent staining of

viable cells can be used to differentiate

live from dead cells

Detection of C Michiganensis subsp Michiganensis in tomato seed extractsDetection of X Axonopodis pv Dieffenbachiae causal agent of anthurium blight (Alvarez etal 1999)Determine viability of R Solanacearum in seed potatoes (van derwolf etal 2004)

47

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

HYBRIDIZATION BASED METHODS

48

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

49

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

Southern blotting

bull Professor Sir Edwin Southern

developed this method in 1975

bull This method Involves separation

transfer and hybridization

bull Detection of a specific DNA

sequence in DNA samples

bull Combines agarose gel

electrophoresis for size

separation of DNA and

hybridization with probeProfessor Sir Edwin Southern

50

51

51

Northern Blotting

Northern blotting is a technique for detection of specific RNA

sequences

Northern blotting was developed by James Alwine and George

Stark at Stanford University (1979)

Electrophoresed RNA is blotted on membrane and hybridized

52

53

Western blotting

bull Western blotting (1981) is an immunoblotting technique which

rely on the specificity of binding between a protein of interest

and a probe (antibody raised against that particular protein) to

allow detection of the protein of interest in a mixture of many

other similar molecules

bull The SDS (Sodium dodecyl sulphate) page technique is a

prerequisite for western blotting

54

Steps in western blotting

Detected

through

auto-

radiography

55

DNA Microarray

56

Microarrays for Rapid Identification of Plant

Viruses

Neil Boonham Jenny Tomlinsonand Rick Mumford

Central Science Laboratory Sand Hutton York YO41 1LZ

United Kingdom

57

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

53

Western blotting

bull Western blotting (1981) is an immunoblotting technique which

rely on the specificity of binding between a protein of interest

and a probe (antibody raised against that particular protein) to

allow detection of the protein of interest in a mixture of many

other similar molecules

bull The SDS (Sodium dodecyl sulphate) page technique is a

prerequisite for western blotting

54

Steps in western blotting

Detected

through

auto-

radiography

55

DNA Microarray

56

Microarrays for Rapid Identification of Plant

Viruses

Neil Boonham Jenny Tomlinsonand Rick Mumford

Central Science Laboratory Sand Hutton York YO41 1LZ

United Kingdom

57

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Western blotting

bull Western blotting (1981) is an immunoblotting technique which

rely on the specificity of binding between a protein of interest

and a probe (antibody raised against that particular protein) to

allow detection of the protein of interest in a mixture of many

other similar molecules

bull The SDS (Sodium dodecyl sulphate) page technique is a

prerequisite for western blotting

54

Steps in western blotting

Detected

through

auto-

radiography

55

DNA Microarray

56

Microarrays for Rapid Identification of Plant

Viruses

Neil Boonham Jenny Tomlinsonand Rick Mumford

Central Science Laboratory Sand Hutton York YO41 1LZ

United Kingdom

57

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Steps in western blotting

Detected

through

auto-

radiography

55

DNA Microarray

56

Microarrays for Rapid Identification of Plant

Viruses

Neil Boonham Jenny Tomlinsonand Rick Mumford

Central Science Laboratory Sand Hutton York YO41 1LZ

United Kingdom

57

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

DNA Microarray

56

Microarrays for Rapid Identification of Plant

Viruses

Neil Boonham Jenny Tomlinsonand Rick Mumford

Central Science Laboratory Sand Hutton York YO41 1LZ

United Kingdom

57

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Microarrays for Rapid Identification of Plant

Viruses

Neil Boonham Jenny Tomlinsonand Rick Mumford

Central Science Laboratory Sand Hutton York YO41 1LZ

United Kingdom

57

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Boonham et al 2007

A schematic diagram detailing a simple approach to virus

detection using a microarray58

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

A microarray designed to detect and

discriminate a range of small spherical

viruses Eg Broad bean wilt virus 2

Indicator host Chenopodium quinoaA small spherical virus was identified

using electron microscopy

Boonham et al 200759

Positive control spots

Detection of virus

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Sensitivity comparison between ELISA and microarray

Boonham et al 2003

Dilution end

point

Histogram showing localbackground fluorescence for the11600 dilution of RNA

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

BIOCHEMICAL METHODS

bull FAME analysis

bull BIOLOG

bull Volatile compound

Biochemical techniques

61

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

What is a BIOLOG

First and only bacterial identification system to identify both gram positive

and gram negative bacteria with a single universal test kit

Add cells

96 wells contains different carbon sources and other test If the cells are metabolically active they reduce the redox dye and a purple colour is formed in al the

positive well

62

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Fatty Acid Methyl Ester ( FAME ) analysis

Change in the fatty acid profile represent a change in the

microbial population

63

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Detection of Diseased Plants by Analysis of Volatile

Organic Compound Emission

RMC Jansen J Wildt IF Kappers

HJ Bouwmeester JW Hofstee

and EJ van Henten

64

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Emission of volatile organic compounds (VOCs)

from non-infected and Botrytis cinereandashinfected

tomato plants

Jansen et al 2011 65

Damaged cell membranes

Local emission of several lipoxygenase (LOX)

oxidative cleavage of C18fatty acids(oxygen and lipoxygenases )

Characterize diseases due to release of VOCs

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Surface Plasmon Resonance (SPR)

The Surface Plasmon Resonance

(SPR) sensor is used for label free

detection and real-time monitoring

Their simplicity and sensitivity make biosensors an effective means of disease diagnosis and monitoring

In SPR technique shift of the resonance angle is observed when the intended species is captured by the immobilized antibody on the sensor surface

66Is it the specific protein the virus fragment or the virion itself

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Development of Surface Plasmon Resonance (SPR) Based Immuno-

Sensing System for Detection of Fungal Teliospores of Karnal Bunt

(Tilletia Indica) a Quarantined Disease of WheatSadhna Singh1 Manoj Singh1 Gohar Taj1 Sanjay Gupta2 and Anil Kumar1

1Department of Molecular Biology amp Genetic Engineering College of Basic Sciences

amp Humanities G B Pant University of Agriculture amp Technology Pantnagar

Uttaranchal India2Department of Biotechnology SBS PG Institute of Biomedical Sciences Balawala

Dehradun Uttarakhand India

Journal of Biosensors amp

Bioelectronics

67

Experiment conducted Interaction of teliosporic wall

antigen with the anti-teliosporic antibody immobilized on sensor

chip

The interaction of antigen at a concentration of 80 40 20 10 50 25

125 0625 0312 0156 078 and 0039 ngμl with immobilized antibody

on sensor chip was examined

Observation

bull The responses increased in proportion to the concentration of teliosporic

antigen due to the change of the refractive index near the SPR sensor chip

68

SPR sensor response after the interaction of different concentrations of antigen over

the immobilized antibody (1 500)

Singh et al 2012

(a) 0312

ngμl

(b)125 ngμl

(c) 5 ngμl

(d) 20 ngμl

(e) 80 ngμl

Advantages of SPR

Major advantages

bull Rapid real-time

bull Non-labeling analysis

bull Miniaturization for portable application

70

71

Phytophthora and Pythium Test Kits 05 per cent of a

plant roots are infected

Tests for Phytophthora Pythium and Rhizoctonia root and crown decay

fungi can be performed on-site by growers in about 10 minutes

(A) Collect and grind samples using abrasive pads

(B) Fold pads and insert them into the extraction solution

(C) Apply solutions to detector

(D) Examine detector dots for color change72

Molecular methods for detection of plant

pathogensmdashWhat is the future

Strategies are needed on how to exploit deduced genomics and

proteomics supported by in silico analysis for establishing

rational disease control measures

The reliability of each specific on-the-spot diagnostic method

needs to be validated before results are used exclusively to

implement costly disease control strategies andor regulatory

actions

73

74

75

SPR sensor response after the interaction of different concentrations of antigen over

the immobilized antibody (1 500)

Singh et al 2012

(a) 0312

ngμl

(b)125 ngμl

(c) 5 ngμl

(d) 20 ngμl

(e) 80 ngμl

Advantages of SPR

Major advantages

bull Rapid real-time

bull Non-labeling analysis

bull Miniaturization for portable application

70

71

Phytophthora and Pythium Test Kits 05 per cent of a

plant roots are infected

Tests for Phytophthora Pythium and Rhizoctonia root and crown decay

fungi can be performed on-site by growers in about 10 minutes

(A) Collect and grind samples using abrasive pads

(B) Fold pads and insert them into the extraction solution

(C) Apply solutions to detector

(D) Examine detector dots for color change72

Molecular methods for detection of plant

pathogensmdashWhat is the future

Strategies are needed on how to exploit deduced genomics and

proteomics supported by in silico analysis for establishing

rational disease control measures

The reliability of each specific on-the-spot diagnostic method

needs to be validated before results are used exclusively to

implement costly disease control strategies andor regulatory

actions

73

74

75

Advantages of SPR

Major advantages

bull Rapid real-time

bull Non-labeling analysis

bull Miniaturization for portable application

70

71

Phytophthora and Pythium Test Kits 05 per cent of a

plant roots are infected

Tests for Phytophthora Pythium and Rhizoctonia root and crown decay

fungi can be performed on-site by growers in about 10 minutes

(A) Collect and grind samples using abrasive pads

(B) Fold pads and insert them into the extraction solution

(C) Apply solutions to detector

(D) Examine detector dots for color change72

Molecular methods for detection of plant

pathogensmdashWhat is the future

Strategies are needed on how to exploit deduced genomics and

proteomics supported by in silico analysis for establishing

rational disease control measures

The reliability of each specific on-the-spot diagnostic method

needs to be validated before results are used exclusively to

implement costly disease control strategies andor regulatory

actions

73

74

75

71

Phytophthora and Pythium Test Kits 05 per cent of a

plant roots are infected

Tests for Phytophthora Pythium and Rhizoctonia root and crown decay

fungi can be performed on-site by growers in about 10 minutes

(A) Collect and grind samples using abrasive pads

(B) Fold pads and insert them into the extraction solution

(C) Apply solutions to detector

(D) Examine detector dots for color change72

Molecular methods for detection of plant

pathogensmdashWhat is the future

Strategies are needed on how to exploit deduced genomics and

proteomics supported by in silico analysis for establishing

rational disease control measures

The reliability of each specific on-the-spot diagnostic method

needs to be validated before results are used exclusively to

implement costly disease control strategies andor regulatory

actions

73

74

75

Phytophthora and Pythium Test Kits 05 per cent of a

plant roots are infected

Tests for Phytophthora Pythium and Rhizoctonia root and crown decay

fungi can be performed on-site by growers in about 10 minutes

(A) Collect and grind samples using abrasive pads

(B) Fold pads and insert them into the extraction solution

(C) Apply solutions to detector

(D) Examine detector dots for color change72

Molecular methods for detection of plant

pathogensmdashWhat is the future

Strategies are needed on how to exploit deduced genomics and

proteomics supported by in silico analysis for establishing

rational disease control measures

The reliability of each specific on-the-spot diagnostic method

needs to be validated before results are used exclusively to

implement costly disease control strategies andor regulatory

actions

73

74

75

Molecular methods for detection of plant

pathogensmdashWhat is the future

Strategies are needed on how to exploit deduced genomics and

proteomics supported by in silico analysis for establishing

rational disease control measures

The reliability of each specific on-the-spot diagnostic method

needs to be validated before results are used exclusively to

implement costly disease control strategies andor regulatory

actions

73

74

75

74

75

75