Multiplex RT-PCR:The Factors Affected Simultaneous Detection of Potato Viruses

A multiplex reverse transcription polymerase chain reaction(m-RT-PCR) was developed for the simultaneous detection of potato virus X, potato virus A and potato virus S. The primers for PVX (562bp) and PVS (182bp) fragments were designed based on the coat protein region and primers for PVA(255bp) based on the P1 gene region. The factors of reagent concentrations, reaction conditions at reverse transcription stage (RT) and reverse transcription-polymerase chain reaction (PCR) stage to the dectection were evaluated. The result showed that the dNTPs concentration, antisense primers ratios of three viruses at RT stage had a great impact on the detection performance , others were the MgCl 2 concentration and annealing temperature at PCR stage, While the reverse transcription time and PCR reaction condition had very little effect to the detection results.