One-step multiplex DiRT-PCR for PLRV, PVY, PVM, PVS, PVA and PVX ready for routine testing directly on tuber sap

Abstract

Potato viruses PLRV, PVY, PVM, PVA, PVX and PVS can cause up to 90% loss of potato harvest. Therefore, they are monitored by law in many countries using DAS-ELISA or conventional real-time RT-qPCR. Previously, we developed a multiplex real-time DiRT-PCR (Direct reverse transcript – polymerase chain reaction), which works directly on diluted tuber sap and thus saves time and chemical processing for RNA extraction or time and space in the glasshouse. So far, this method only ran on sap of single tubers which is not practical for routine testing. We are now able to sensitively test for the presence of six viruses in two multiplex reactions using the real-time DiRT-PCR on pooled samples of ten tubers. Here we show that there is an “almost perfect” agreement (Gwet’s AC1 index) comparing this multiplex real-time DiRT-PCR on pooled samples with DAS-ELISA and a commercial RT-qPCR kit with a rapid extraction method. The multiplex real-time DiRT-PCR is now ready to be used for routine testing.