techniques for the detection of seed transmitted plant viruses
TRANSCRIPT
TECHNIQUES FOR THE DETECTION OF SEED TRANSMITTED PLANT VIRUSES
By: Preeti Rana
M.Sc. Biotechnology – I Year
Introduction
Viruses Seed Transmitted Viruses Virus diagnosis
Diagnostic Techniques
Biological assays
~ Symptomatology - Indicator plants
- Host range
Serological methods Electron microscopy Nucleic acid based methods ~ PCR
~ Nucleic acid hybridization methods
Biological assays
Symptomatology and host range
- symptoms are the initial steep in disease diagnosis
- some symptoms indicates the infecting virus belongs to particular group
Indicator plants - e.g.: Chenopodium, Nicotiana, Phaseolus, Vigna
Sedimentation properties Cytopathology
Symptoms
Yellow mosaic symptoms on lettuceCaused by lettuce mosaic virus
Yellow vein-banding -caused byGrapevine fanleaf virus
Serological Techniques
ELISA*
- Double antibody sandwich (DAS-ELISA)
- Triple antibody sandwich (TAS-ELISA)
- Direct antigen coating (DAC-ELISA)
- Penicillinase based (PNC-ELISA)
Dot Immunobinding Assay (DIBA) Tissue Blotting Immunoassay (TBIA) *Enzyme linked immunosorbent assay
Other serological techniques
Precipitin reaction Immunodiffusion tests
- radial diffusion
- gel double diffusion
Agglutination tests - slide agglutination
- latex particle test
- haemoagglutination tests
- virobacterial agglutination tests
Immunosorbent electron microscopy (ISEM)
Electron microscopy
Round and smooth - isometric viruses e.g. cucumovirus Round and knobbly - e.g. tombus and tomyvirus Ovoid and spherical – e.g. ilaviruses Angular – e.g. Nepo and comovirus Bullet shaped – Rhabdovirus group
Nucleic acid based
Nucleic acid spot hybridisation (NASH) - target nucleic acid is immobilized on nitrocellulose sheet
Polymerase chain reaction (PCR) - able to detect pathogens by targeting their genetic material
PCR Formats
RT-PCR -for RNA viruses IC-PCR -for DNA & RNA viruses Real time PCR -for RNA viruses
Immunocapture PCR combines the advantages of serology and PCR into a very sensitive method of detection
Multiplex PCR allows for simultaneous and sensitive detection of different DNA/RNA in a single reaction
Real time PCR combines application, detection and quantification in a single step
PCR-ELISA -for viruses of fruit, nutshells, grapevine etc.
Microarrays
DNA microarrays or biochips are made of a surface on which multiple capture of DNA sequence of the targets is possible.
Purpose is to detect numerous sequences in a single assay
Used at research level
Summary
Sensitivity, specificity and appropriateness.
Cost and labour Nucleic acid hybridization or serology All techniques need validation before
use in routine testing