techniques for the detection of seed transmitted plant viruses

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