The overall purpose of this research was to develop a reverse transcription (RT) polymerase chain reaction (PCR)-based method for detection of viruses in infected potato plant. Potato virus A (PVA), Potato virus X (PVX), Potato virus Y (PVY) and Potato leafroll virus (PLRV) are four different virus species of the most important viruses infecting cultivated potatoes. RT PCR-based methods for detecting the four potato viruses were successfully developed in this study.
A total of 1,120 symptomatic leaf samples from field and from greenhouse were collected from December 2007 to July 2009 in three Northern provinces, Chiang Mai, Chiang Rai and Tak, of Thailand. Samples of symptomatic plants were analyzed for virus infection by RT-PCR using specific oligonucleotide primers. In the first year of this research project (Oct 2007 – Sep 2008), optimization and application of a reverse transcription-polymerase chain reaction (RT-PCR) system have been studied for detection of five potato viruses. A nucleic acids extraction protocol was developed for both potato-infecting DNA and RNA viruses. By using an oligo(dT) as a common primer for RNA templates containing a poly(A) tract, a RT-PCR system have been developed for detection of four potato viruses (PVA, PVS, PVX and PVY) which contained a poly(A) tract at their 3’-end. In the second year (Oct 2008 – Sep 2009), three viral genera including four potato viruses, potyvirus (PVA and PVY), potexvirus (PVX) and polerovirus (PLRV) were used to develop manifold systems of RT-PCR. Several specific or degenerate oligonucleotide primers were designed on aligned virus sequences published in the GenBank covering the genome of different PVA, PVX, PVY and PLRV isolates, consensus regions were identified. These designed primers were used as either forward or reverse primers in PCR which were tested for their specificity and sensitivity. Four primer pairs, A05F/A04R, X01F/X02R, Y02F/Y03R and R38F/R32R, were preferred and used as a set of virus species-specific primer in PCR for development of RT-PCR methods for detecting PVA, PVX, PVY and PLRV, respectively. Furthermore, merging an oligo(dT) primer with the ‘R32R’ specific reverse primer can be used to simultaneously prime RT of both polyadenylated and non-polyadenylated RNAs, and PCR for simultaneous detection of two different viruses, PVY and PLRV with a mixture of primers Y02F, Y03R, R38F and R32R, is reported.