molecular techniques and applications in...

Molecular Techniques and

Applications in Bacteriology

DR. M. Talebi

Assistant Professor

Iran Medical University

Molecular Microbiology

• Microbiologists have always searched and continue to search for more rapid and efficient ways to detect and characterize microorganisms

• The molecular techniques have provided some of the

most powerful tools to date • The migration of modern molecular diagnostic

methods from the basic science research laboratories into clinical laboratories has been underway for more than a decade

Molecular Microbiology

• These assays have revolutionized the microbiology laboratory

and have changed the way we detect, characterize and quantify

microorganisms directly in clinical specimens and from

cultures

• Over the past 15 years, these techniques have progressed from

once cumbersome, highly technical, labor-intensive assays to

user-friendly tests that are so rapid that it is, in some instances,

possible to achieve test results within an hour from the start of

the assay

Molecular Diagnostics

• Fastest growing area in laboratory medicine

– Detection technologies

– Commercial detection kits

– Analyte specific reagents

– Instrumentation options

Molecular Diagnostics

- Diagnosis of infectious diseases

- Genetic identification

- Diagnosis of genetic diseases

Microbial detection

• Traditional methods time-consuming: Culture

media, followed by Isolation, Biochemical

Identification, and sometimes Serology

• Recent advances in technology make detection

and identification Faster, more Convenient,

more Sensitive, and more Specific

Leading uses for nucleic acid based

tests • Nonculturable agents

– Human papilloma virus

– Hepatitis B virus

• Fastidious, slow-growing agents

– Mycobacterium tuberculosis

– Legionella pneumophilia

• Highly infectious agents that are dangerous to culture

– Francisella tularensis

– Brucella species

– Coccidioidis immitis

Leading uses for nucleic acid based

tests

• In situ detection of infectious agents

– Helicobacter pylori

– Toxoplasma gondii

• Agents present in low numbers

– HIV in antibody negative patients

– CMV in transplanted organs

• Organisms present in small volume specimens

Leading uses for nucleic acid based

tests

• Differentiation of antigenically similar

agents

– May be important for detecting specific virus

genotypes associated with human cancers

(Papilloma viruses)

• Antibiotic resistance genes

• Non-viable organisms

– Organisms tied up in immune complexes

What are the advantages of using a molecular test?

• High sensitivity – Can theoretically detect the presence of a single organism

• High specificity – Can detect specific genotypes

– Can determine drug resistance

– Can predict virulence

• High transport toleration

• Speed – Quicker than traditional culturing for certain organisms

• Simplicity – Some assays are now automated

What are the advantages of using a molecular test?

• Need an accurate and timely diagnosis

– Important for initiating the proper treatment

– Important for preventing the spread of a contagious disease

Diagnostic methods for detection of Chlamydia trachomatis -

the most common STD in Europe

WHO: - sensitivity > 90% specificity > 99%

- serology – for scientific purpose only

Method Sensitivity Specificity Price(€) Comments

Cell culture 40-85% 100% 6,3 Low sensitivity

Immunofl. 50-90% 85-95% 5,2 Needs expertise

Gen-Probe 70-85% 95-97% 10,9 Low sensitivity

PCR 70-95% 97-100% 10,4 Contamination

Review: The structure of DNA

Antiparallel Strands

Unzipping

Structure of DNA

• DNA deoxyribonucleic acid

– Two long polynucleotide chains

with 4 nucleotide subunits

• Nucleotides subunits

– Sugar, phosphate groups, base

– Adenosine (A), Guanine (G),

Cytosine (C), Thymine (T)

• “Backbone”

– Covalently linked nucleotides

• Hydrogen bonds hold two chains

together

Nucleotide

Deoxyribonucleic Acid

DNA Structure – Double Helix

• Single stranded DNA will form

double stranded DNA only with it’s

complement

• Polarity – 3’ (3’ hydroxyl) and 5’ (5’

phosphate)

• Complementary base pairing G-C

and T-A

• Hydrogen Bonding holds strands

together

DNA Replication

DNA synthesis

• Remember that DNA replication is semiconservative:

Function of RNA-Information Transfer

READING (TRANSCRIPTION) AND INTERPRETATION

(TRANSLATION) OF THE GENETIC CODE

Molecular diagnostics – how it works?

• Every organism contains some unique,

species specific DNA sequences

• Molecular diagnostics makes the species

specific DNA visible

Different types of nucleic acid

molecular techniques

What are the different types of nucleic acid molecular techniques that are used?

• Direct probe testing – better for identification than for detection because it is not as sensitive as amplification methods

• Amplification methods – used to improve the sensitivity of the nucleic acid testing technique

– Target amplification

– Probe amplification

– Signal amplification

– Combinations of the above

Direct probe testing

• Hybridization – to come together through complementary base-pairing.

– Can be used in identification.

– In colony hybridization the colony is treated to release the nucleic acid which is then denatured to single strands

• Labeled single-stranded DNA (a probe) unique to the organism you are testing for is added and hybridization is allowed to occur

• Unbound probe is washed away and the presence of bound probe is determined by the presence of the label.

In-Situ Hybridization

• Target nucleic acid found in intact cells

• Provides information about presence of specific DNA targets and distribution in tissues

• Probes must be small enough to reach nucleic acid

• Radioactive or fluorescent tags used

Fluorescent In-Situ Hibridization FISH

Hybrid Capture

CLINICAL APPLICATIONS

• As a rapid and specific method of identifying microorganisms in culture

• PACE products – N. gonorrhoeae

– C. trachomatis

– Group A Streptococcus

– Mycobacteria

– Dimorphic fungi

– Histoplasma capsulatum

– Blastomyces dermatitidis

– Coccidioides

– And………

• Genetically encoded virulence factors in culture isolates and specimens, e.g. detection of Shiga-like toxins in Escherichia coli O157

• These assays, when performed from cultures, have sensitivities and specificities in the high 90s to 100%. The advantage of using these assays is time savings.

Target amplification

PCR

How PCR Works

Step 1 - Denaturation (optimal temperature is 94°C)

By heating the DNA, the double strand melts and open to single stranded DNA.

How PCR Works

Step 2 - Annealing (optimal temperature is 60°C)

Add single stranded primers that match to sequences of the target DNA. The single-stranded primers bind to their complementary single-stranded bases on the denaturated DNA.

How PCR Works

Step 3 - Extension (72°C is the ideal temperature)

Add single DNA nucleotides and an enzyme that reads opposing strains sequence and extend primers sequence to match (complementary). Taq polymerase is used because it can withstand high temperature necessary for DNA strand separation and can be left in reaction to attach and start copying the template. The result is two new helixes in place of the first.

Replication – Two exact copies of the original DNA are created. The process can be repeated again and again to achieve suitable levels of detection. Since both the old and new DNA can be copied the amplification grows

How PCR Works

•DNA heated – paired strands separate

• Large excess of primers added, some are complementary to sequences on target DNA. Cool the reaction mixture to allow binding to take place.

• Add single DNA nucleotides and enzyme that reads opposing strains sequence and extend primers sequence to match (complementary).

• Enzyme synthesizes new DNA. Process is repeated both new DNA and old are amplified

Review

36

PCR laboratory

Sample handling

DNA preparation

Clean room

Stock solutions

Laboratory

Mixing site

Thermocycler

Amplification

Detection

Documentation

QC & QA Quality control & assurance

R & D (Research and development)

Alternatives: - commercial kits

- robots + kits

No alternative

Hard choice - what is the best

• In-house (home-brew)

• PCR or commercial PCR kits

PCR Reaction Components

• Water

• Buffer

• DNA template

• Primers

• Nucleotides

• Mg++ ions

• DNA Polymerase

• Extras

PCR components

Needs accurate

temperature control

PCR machines

Automatic cycling of

temperature

Buffer

• Each DNA polymerase works best under optimal temperature, pH and salt concentration

• PCR buffer provides optimal pH and salt condition • Buffer

– Stabilizes the DNA polymerase, DNA, and nucleotides – 500 mM KCl – 100 mM Tris-HCl, pH 8.3 – Triton X-100 or Tween

• Must match polymerase • Can vary over a slight range

– Not much difference in range from 0.8 X to 2.0 X – Primer efficiency reduced outside this range

• Required for DNA polymerase activity

• Essential co-factor of DNA polymerase

• Stabilizes the DNA double-helix

• Mg ions form complexes with dNTPs and primers

Mg2+ ions

• Too little • Enzyme won’t work • A low yield of PCR product

• Too much • DNA extra stable • Non-specific priming • Band smearing • Increase the yield of non-specific products

Used at 0.5 to 3.5 uM in the assay

Mg2+ ions

PCR Reaction: Nucleotides

• 20-400 uM works well

–dATP, dGTP, dCTP, dTTP

– Stored at 10mM, pH 7.0

–Too much: can lead to miss priming and errors

–Too much: can scavenge Mg++

–Too low: faint products

PCR Reaction: Primers

• What are primers? – Short artificial DNA sequences

• Match template DNA • Polymerase can only start synthesizing from double stranded DNA • Start where primer anneal

• Primers – Specific for ends of amplified region – Forward and Reverse – Annealing temps should be known

• Number of freeze-thaws • Contamination • Amount

– 100-500 nM typical – Too low: low amplification – Too high: low amplification

Primers

• Where do we get primer sequences from?

• Check databases

• Freely available on internet (GenBank)

–Results not publishable without primer information

– Primer design from published sequences

• Design primers in conserved regions

Primer Design Criteria

1. Uniqueness: ensure correct priming site

2. Length: 17-28 bases. This range varies

3. Base composition: average (G+C) content around 50-

60%; avoid long (A+T) and (G+C) rich region if possible

4. Optimize base pairing: it’s critical that the stability at 5’

end be high and the stability at 3’ end be relatively low to

minimize false priming.

Primer design

• Primer pairs should have similar annealing temp

– length, %GC content

– Tm = 4(G + C) + 2(A + T) oC.

• Minimal (<3bp) between-primer-complementarity

• Primers should have no self complementarity

5’-ACTGTGCCATAGATGCAG-3’ ||||

3’-CAACTGCACCGTATGCAT-5’

5’-ACTGT AGAT-3

GCC ATA G

GC

• Programs on the web to design primers

– Links on webpage

There are a bunch of good PCR primer design

programs on the web:

Primer 3 at the MIT Whitehead Institute

http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

Cassandra at the Univ. of Southern California

http://www-hto.usc.edu/software/procrustes/cassandra/cass_frm.html

GeneFisher by Folker Meyer & Chris Schleiermacher

at Bielefeld University, Germany

http://bibiserv.TechFak.Uni-Bielefeld.DE/genefisher/

Xprimer at the Virtual Genome Center, Univ. Minnesota

Medical School

http://alces.med.umn.edu/rawprimer.html

Primer Design on the Web

Primer3

http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

Primer3

Primer3

DNA Polymerase

• Matches buffer

• Concentration: Typically 0.5 to 1.0 U/rxn

• Taq, Vent, Pfu, others

• Native or Cloned

• 3’-5’ Exo nuclease – proofreading

• Fidelity (Error Rate)

– Taq 1/10,000nt, Pfu 1/1,000,000

• Processivity

PCR Reaction: Template DNA

• DNA template

– Contains region to be amplified

– Any DNA desired

– Should be free of polymerase inhibitor

• Degradation

• Contamination

• Purity

– Interfering factors, eg. enzymes, salts

• Presence of “poisons”

– Eg. EDTA which scavenges Mg++

PCR Cycling Parameters

• Denaturation Temp

• Annealing Temp

• Extension Temp

• Time

• Number of Cycles

• Reaction Volume

PCR Cycling Parameters

• Reaction Volume

• Doesn’t affect PCR results

• 25ul, 50ul, 100ul all work.

PCR optimization - rules

• Maximize stringency

– Highest annealing temp

– Lowest MgCl2

• Minimize number of cycles

– Taq degradation

– Production of non-specifics

– Taq errors

• Most significant parameters

– Annealing temperature

– MgCl2

Common PCR Problems

• Contamination

• No or weak product

• Primer dimers

• Non-specific products

PCR - in practice

• You are never setting up only a single PCR

reaction

– Make up master mix

• Buffer, primers, MgCl2, water, dNTPs, Taq

– When calculating master mix volume, add a bit (~1

sample’s worth) extra to allow for pipetting errors

• Negative control

– No template DNA

• Check for contamination

• Positive control

– Something you know works

Polymerase chain reaction

The benefits of PCR based diagnostic testing

Rapid diagnosis Detection

Same day result

High accuracy

high specifity

high sensitivity

Modifications of PCR

Multiplex PCR: What

• PCR using several primer pairs

SIMULTANEOUSLY

• Detect several genes at once

• Typically generates a product band for

each primer pair

• Same as regular PCR

• Care in primer design – Much greater chance of primer-dimers

– Annealing temperatures must be close

Multiplex applications

• Detection of multiple organism simultaneously

– Meningitidis

– Sepsis

– Respiratory infections

– Urogenital infections

• Detection of a bacteria and antibiotic resistance

• Detection of bacteria and virulence genes

Nested PCR

• The nested PCR has been designed to increase sensitivity and specificity allowing detection of smaller quantities of target DNA

• Nested PCR is a two-step process using two sets of primers

– Conventional PCR does not detect latent human herpes virus 6 (HHV-6) DNA in peripheral blood mononuclear cells

– This is also the case for the detection of Streptococcus pneumoniae in community-acquired pneumonia

Reverse Transcriptase PCR

• Detect specific RNA sequences in samples

• The detection of cDNA by RT-PCR of mRNA encoded by a pathogen may be evidence of active infection

• Conventional PCR cannot distinguish between living and dead organisms

• Detection of viable Mvcobacteria and the effectiveness of antimicrobial therapy

Real-time PCR Principles

Real-time PCR monitors the fluorescence emitted during the reaction as

an indicator of amplicon production at each PCR cycle (in real time) as

opposed to the endpoint detection

* based on the detection and quantitation of a fluorescent

reporter

* the first significant increase in the amount of PCR product

(CT - threshold cycle) correlates to the initial amount of

target template

Real-Time PCR Principles

Three general methods for the quantitative assays:

1. Hydrolysis probes

(TaqMan, Beacons)

2. DNA-binding agents

(SYBR Green)

Log-view augments this part

(www)

The Amplification Plot contains valuable information for the quantitative measurement of DNA or RNA. The Threshold line is the level of detection or the point at which a reaction reaches a

fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is

called the Cycle Threshold, CT. These two values are very important for data analysis using the 5’ nuclease assay.

What is CT?

log view

What is Wrong with Agarose Gels?

* Poor precision

* Low sensitivity

* Low resolution

* Non-automated

* Size-based discrimination only

* Results are not expressed as numbers

* Ethidium bromide staining is not very quantitative

Real-time PCR advantages

* not influenced by non-specific amplification

* amplification can be monitored real-time

* no post-PCR processing of products (high throughput, low contamination risk)

* ultra-rapid cycling (30 minutes to 2 hours)

* requirement of 1000-fold less template than conventional assays

* confirmation of specific amplification by melting curve analysis

* most specific, sensitive and reproducible

Real-time PCR disadvantages

* not ideal for multiplexing

* setting up requires high technical skill and support

* high equipment cost

* * *

Applications

• Warren et al. described a sensitive and specific test for rapid detection of MRSA directly from nasal swab specimens, which is based upon real time PCR using a molecular beacon probe

– The time from sampling to having the result was only 1.5 h

• VRE a multiplex real time PCR assay for the Roche Lightcycler employing FRET hybridization probes, for the simultaneous detection of vanA and vanB

• The FDA-approved screening tests for

– Group A streptococcus

– Bordetella pertussis

– Legionella pneumophila

– Mycoplasma pneumoniae

– Chlamydia pneumophila

Broad-range PCR

• Certain functional domains within the genome (e.g. 16S

ribosomal RNA) that possess phylogenetic information are highly conserved in the prokaryote kingdom.

• This allows amplification of target DNA from virtually all prokaryote organisms with a single set of primers

• Subsequent nucleic acid sequencing of the amplified DNA, and alignment of these sequences against reference sequences (i.e. homologous sequences of 'known' organisms), allows the identification of the pathogen or the possible discovery of a 'new' organism

• Broad-range PCR has successfully identified novel, fastidious and nonculturable agents from human tissue or blood

• The etiology of bacillary angiomatosis (Relman et al., 1990), the unculturable and previously unidentifiable bacillus associated with Whipple's disease, Tropheryma whippli

• Ehrlichia were identified by amplification of 16S ribosomal DNA

• These include the heat shock protein 65 (hsp65) and ttroEL gene to characterize clinical mvcobacterial

Diagnostics of bacterial infections using DNA microarrays

• Which are not yet fully developed for bacterial diagnostics, but will be developed

further in that direction in the future.

– Methicillin-resistant Staphylococcus aureus

– Rapid detection of rifampicine and isoniazide resistance in M. tuberculosis using SNP microarrays

AND Other current and novel methods under development……