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 Yellow vein-banding -caused by

lettuce Grapevine fanleaf virus
Caused by lettuce mosaic 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