This document discusses various nucleic acid-based methods for virus indexing and detection in plants. It describes techniques like RT-PCR, multiplex PCR, immuno-capture PCR, real-time PCR, nucleic acid spot hybridization, and DNA microarray technology that can be used to detect viral genomes from crude or purified plant samples. Reverse transcriptase PCR is commonly used to detect gene expression and identify infections. Multiplex PCR allows simultaneous detection of multiple viruses. Real-time PCR provides reliable and sensitive quantification of plant viruses. Nucleic acid spot hybridization is suitable for testing large numbers of samples for detection of viruses like BBTV. DNA microarrays can detect a wide range of plant viruses and identify new pathogens through hybridization patterns

1. NUCLEI ACID BASED
VIRUS INDEXING METHODS

2. INTRODUCTION
VIRUS INDEXING : A technology for the production of
quality planting material
What does ‘virus-indexed’ mean ?
A virus – indexed variety is one that has been tested
and deemed free of viruses it was tested for up to the point
of testing .

3. VIRAL GENOME BASED METHODS
 RT- PCR
 MULTIPLEX PCR & IMMUNO-CAPTURE PCR
 REAL TIME PCR
 NUCLEIC ACID BASED SPOT HYBRIDIZATION ( NASH)
 DNA MICROARRAY TECHNOLOGY

4. METHOD OF VIRUS INDEXING
1. PHYSICAL ASSAY : Electron microscopy
2. BIOLOGICAL INDEXING : Sap transmission test
3. MOLECULAR INDEXING : Nucleic acid based and
protein based ( Serological assay )

5. REVERSE TRANSCRIPTASE PCR
• RT-PCR : A variant of PCR is a technique commonly used
in molecular biology to detect RNA transcript levels.
• It qualitatively detect gene expression through the
creation of complementary DNA .
• Hence it is frequently used in the expression analysis of
single or multiple genes and the exp. Patterns for
identifying infections and diseases.

6. • PCR based detection
system is highly successful
for all banana viruses
(BBrMV, BBTV , CMV ,
BaMMV )
• Exc : BSV

7. MULTIPLEX PCR & IMMUNO CAPTURE PCR
 Simultaneous detection of viruses
 In IC-PCR , virus particles trapped onto a specific
antibody Disruption nucleic acids released
amplified .
 Especially used in case of low virus titre value .

9. REAL TIME PCR & END POINT PCR
 Quantification of plant viruses
 More reliable , sensitive and specific than PCR .
 Reduce cross contamination.
 Real time PCR : Validate analyses and gene expression
changes on global scale.
 End point PCR : Measures gene expression changes in
small number of samples.

11. NUCLEI ACID SPOT HYBRIDISATION
 Specific hybridization used in viroid
detection.
 Detect nuclei acid in both ss and ds
replicate forms.
 Dot blot hybridization / NASH :
Appropirate technique to test
large number of samples in a short time.

12. • Viral genomes are detected from crude or purified
samples after hybridizing with labelled specific probes.
• Sent to testing centres.
• NRCB has developed NASH technique for BBTV
• Also applied for the detection of BBrMV, CMV and BSV.

13. DNA MICROARRAY TECHNOLOGY
• Detect a myriad of viruses, viroids and phytoplasmas
infecting the plant .
• The unique pattern of hybridization for each of the
pathogen will be the distinguishing feature of this
technique.
• New pathogens could be identified through the use of
microarray sequences derived from highly conserved
regions in a given pathogen group.

14. METHODS OF VIRUS ELIMINATION
 Thermo-therapy and meristem culture
 Cryo-therapy
 Chemo-therapy
 Anti viral antibiotics
 Micrografting

15. SUMMARY
Not all plants produced through a combination of
therapies or by any other methods of virus eradication
programmes are virus-free.
Hence the plants should be indexed several times at
regular intervals during the first 18 months to
completely get rid of viruses.

16. THANK YOU