BP Singh

Techniques for Detection of Plant Viruses 169specifically trapped and decorated with the antiserum and thecontaminants are washed away.d) Complimentary DNA (eDNA) probes: This method is basedon the fact that complimentary strands of nucleic acid hybridizewith one another. It has a potential of detecting extremely lowlevel of inoculum or latent infections in plant/planting materials.Basically the method involves the immobilization of a spotor dot of sap extract from the plant under test on a solid matrixand the detection of viral nucleic acid sequences in that spot byuse of a hybQ"dization probe. For preparing a DNA probe, DNA,complimentary to RNA is synthesized from the RNA templateusing.the enzyme reverse transcriptase. If this DNA is then madedouble stranded it can be cloned in a bacterial plasmid and thusimmortalised. As much qf the DNA copy of the viral sequences,as required, can be prepared and it can also be manipulated usingrecombinant DNA techniques. The probe nucleic acid usuallycarries reporter molecules which can be detected when it ishybridized to the immobilIzed viral sequences. 'e) Polymerase chain reaction (PCR) : This involves rapid andhighly specific in-vitro amplification of selected DNA sequences,for which specific primers are synthesized. The p.roduct obtainedfrom this cycle amplification process is separated by gelelectrophoresis and identified by specific cDNA probes. With itsrelative simplicity and high sensitivity (detecting picogramquantities of viral nucleic acids in infected tissues) PCR method hashigh potential in virus indexing programmes. However, the prerequisiteof having known viral sequences to allow synthesis ofsuitable primers limits its application to well characterised viruses.C) Virus indexing _For poorly characterized or unknown viruses detectionmethods either are not available or are not sensitive. In such casesbioassays using sap and graft inoculation to a range of sensitiveindicators are still the only methods available, . Conclusions basedon negative results in these methods are reliable only when testingis repetitive and includes alternative metho9.s using different virusproperties for detection.