OPTIMIZATION OF DUPLEX RT-PCR FOR SIMULTANEOUS DETECTION OF POTATO VIRUS Y AND S

Abstract

A duplex Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay was developed for simultaneous detection of two viruses infecting potato i.e. Potyvirus (Potato virus Y) and Carlavirus (Potato virus S). Specific primers were designed against coat protein gene of both the viruses (PVY and PVS) and uniplex RT-PCR assay was standardized for detection of these viruses (PVY and PVS) individually. There after, an attempt was made to detect both viruses in a single reaction. RT-PCR conditions were optimized by altering PCR mix i.e. dNTPs, primer and Taq DNA polymerase. This optimized PCR mix and PCR conditions showed an expected size of amplicons in duplex RT-PCR with respect to PVY (380 bp) and PVS (567 bp). Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both the viruses in potato tubers and field infected potato plants. It could detect both viruses in field (naturally) infected plants and tubers. It has the detection sensitivity similar to that of uniplex RT-PCR assay for respective viruses. The duplex RT-PCR tested here provides a simple, rapid, sensitive and convenient way for simultaneous detection of PVY and PVS. In addition, it reduces the time and cost of the consumables and can be used for routine detection of both the viruses simultaneously in seed certification programmes.