P-116. Reverse Transcription Polymerase Chain Reaction (RT-PCR) based Detection of Indian isolates of Potato Virus M

PVM is a mechanically transmitted virus of genus Carlavirus, family Flexiviridae. PVM is wide spread in commercial stocks where ever potatoes are grown. Yield depression of 10–15% has been observed depending upon cultivar and virus strain. In order to have a reliable and sensitive detection, degenerate primers containing ORF and stop codon of coat protein (CP) gene were designed. All the available sequences bordering CP gene were picked up from NCBI database. These sequences were aligned and region of least degeneracy was chosen for primer design. Total RNA from infected leaf tissue was extracted by using Au-Prep Plant RNA kit (Life Tech Inc.). cDNA was synthesized by using random hexamer primers and 20U MuLV reverse transcriptase enzyme. The PCR reaction was optimized. The expected PCR product of about 950 bp size was obtained. To confirm authenticity of the PCR product, it was cloned in pDrive vector (Qiagen Inc.) and cloning confirmed by restriction digestion and colony PCR. Further, specificity of the clone was determined by nucleic acid spot hybridization of run-away transcripts with viral RNA. The reaction conditions were optimized at initial denaturation of 95°C for 4 min followed by 35 cycles each of 1 min at 95°C, 1 min at 58°C and 1min at 72°C the final extension was given for 10 min at 72°C. The protocol was validated by testing in-vitro germplasm accessions and field samples and comparing the results with those of ELISA and IEM. The results of RT-PCR were at par with those of IEM but better than those of ELISA.