Simultaneous detection of several important viruses in naturally infected potato plants using multiplex RT-PCR in comparing with ELISA

Potato virus Y (PVY), Potato virus X (PVX), Potato virus S (PVS) and Potato leaf roll virus(PLRV), are the most important viruses of potato and can cause serious diseases andsignificantly reduce the yield and quality of potato crops. The incidence of these viruses isreported from almost all main potato growing regions in Iran. Therefore, application of a sensitive, rapid and inexpensive procedure for simultaneous detection of potato viruses is very important for management of their control. In the present research, a sensitive and reliable multiplex RT-PCR technique for the detection of PVY, PVX, PVS, PLRV and 18S rRNA as internal control was employed and compared with ELISA, the method used routinely to assay potatoes for virus infections. Total RNA was extracted from virus infected potato leaves. Then cDNA for above viruses and 18S rRNA was individually synthesized using their specific reverse primers and single RT-PCR for each virus and 18S rRNA. Subsequently, multiplex RT-PCR was carried out with five primer pairs in a single reaction. As a result, five different fragments (145-342 bp) specific to the viruses and the internal control were simultaneously amplified and were identified on the basis of their molecular sizes. The sensitivity of our optimized system also was compared with that of commercial DAS-ELISA for detection of PVY, PVS and PLRV. The results showed that the sensitivity of multiplex RT-PCR was 100-fold greater for detection of PVY, PVS and PLRV and the duplex RT-PCR was 1000-fold greater for detection of PVY than that of commercial DASELISA.