A multiplex RT-PCR for the detection of Potato yellow vein virus, Tobacco rattle virus and Tomato infectious chlorosis virus in potato with a plant internal amplification control

Abstract

A multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed using published primers to simultaneously detect three viruses infecting potato (Potato yellow vein virus, Tomato infectious chlorosis virus and Tobacco rattle virus), including an internal amplification control. The characteristics of the published primers were analysed to see if they could be used for multiplex RT-PCR. Amplification of the three target viruses and a plant internal control was then optimized by adding bovine serum albumin during cDNA synthesis, increasing the PCR extension time, reducing the PCR extension temperature and optimizing the concentration of each pair of primers. This optimization produced a multiplex assay with a detection sensitivity of the same order as simplex RT-PCR. This approach provides a simple and convenient way to develop multiplex assays using published primers directly rather than designing new primers. The multiplex RT-PCR is a sensitive and cost-effective method for detecting multiple viruses in potato and can be used for quarantine and certification programmes. © 2008 BSPP.