A MULTIPLEX RT-PCR ASSAY FOR SIMULTANEOUS DETECTION OF FIVE VIRUSES IN POTATO

Potato viruses including Potato aucuba mosaic virus (PAMV), Potato leafroll virus (PLRV), Potato virus M (PVM), Potato virus S (PVS) and Potato virus X (PVX) are well known viruses infecting potato and can cause serious economic losses. In this study, a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed and standardized for simultaneous detection of PAMV, PLRV, PVM, PVS and PVX with elongation factor 1-alpha (ef1 alpha) as plant internal control. At least three primer pairs were designed within the conserved regions of genomic sequences of five potato viruses and ef1 alpha of potato for single and/or multiplex RT-PCR. The reaction conditions were initially optimized by selecting primer pairs and standardized the individual RT-PCR. Then, one pair of primer for each virus including ef1 alpha was selected for multiplex RT-PCR producing distinct fragments of 305, 452, 408, 363, 565, 803 bp, representing PAMV, PLRV, PVM, PVS, PVX and ef1 alpha, respectively. All the primers were designed based on specificity and compatibility and the reliability was confirmed by cloning and sequencing followed by BLAST analysis. The sensitivity of single as well as multiplex RT-PCR was evaluated. The assay could detect all five viruses including ef1 alpha even at total RNA dilutions of 10(-3) and total cDNA dilution of 10(-7). The multiplex RT-PCR assay was then used to test virus infections from field samples of potato (leaves and tubers) collected from different states of India along with plant internal control. This multiplex RT-PCR was equally enough in detecting mixed infection of five viruses in foliage as well as tubers. The results indicate the robustness and reliability of the assay for virus indexing of mother stocks for five viruses simultaneously in potato seed production and will be useful for seed certification programme, breeding and epidemiological studies.