Simultaneous detection of potato viruses A and M using CP gene specific primers in an optimized duplex RT-PCR

Abstract

A duplex reverse transcription PCR (RT-PCR) assay was developed for simultaneous detection of a potyvirus (Potato virus A; PVA) and a carlavirus (Potato virus M; PVM) infecting potato crop. Six coat protein (CP) gene specific primer pairs (three each for PVA and PVM, respectively) were designed for both the viruses. Among these, primers PVACPF3/ PVACPR3 and PVMCPF2/PVMCPR2 successfully co-amplified the CP genes of both viruses at an annealing temperature of 54 °C. Primer PVACPF3/ PVACPR3 produced an amplicon of 560 bp for PVA and primer PVMCPF2/PVMCPR2 produced an amplicon of 408 bp for PVM in the same reaction. The present study has demonstrated for the first time that CP gene specific primers can be used to detect PVA and PVM simultaneously in an optimized RT-PCR. These primer pairs and a duplex RT-PCR optimized in the present study offers a reliable diagnostic tool for simultaneous detection of PVA and PVM in infected plant leaves and quarantine programmes.