Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses

Multiplex PCR is an important technique for detecting a variety of pathogens simultaneously in a single assay. Previous research has focused on optimising the factors affecting reliable multiplex PCR, including primer design, PCR components and conditions, and inhibitors in samples. In this study, the interaction of primers to form complex secondary structures including visible dimers and invisible “primer clusters”, a novel form of primer secondary structure found during this research, were shown to be the most important factors affecting successful multiplex PCR. Approaches to mitigate primer interaction and eliminate inhibitors were tested, including: reduction of primer concentrations especially those with preferential amplification; decrease of PCR extension temperature; increase of extension time and PCR cycles; and addition of bovine serum albumin. Based on these approaches, a multiplex RT-PCR with sensitivity comparable to the simplex PCR for individual viruses was developed for the detection of Raspberry ringspot virus, Strawberry latent ringspot virus and Tomato bushy stunt virus. A plant internal amplification control was also included. These approaches may be useful as a guideline for the development of multiplex PCR protocols for the detection of other pathogens or organisms associated with plants, humans, animals and the environment.