Optimization and Application of a Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses Using 18S rRNA as an Internal Control

Abstract

Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.