Development of a simple one-step multiplex RT-PCR system for simultaneous detection of DNA and RNA viruses of cucurbit leaf crumple virus, cucurbit yellow stunting disorder virus, squash vein yellowing virus, and cucurbit chlorotic yellows virus

A highly sensitive one-step multiplex RT-PCR (OMRT-PCR) system with an internal plant gene control was developed to simultaneously detect single and mixed RNA DNA viral pathogens affecting watermelon squash in the Southeastern United States. Current methods rely on a singleplex detection for several viruses which is time-consuming large-scale field monitoring. The gene-specific primers one virus, cucurbit leaf crumple virus (CuLCrV), three viruses, yellow stunting disorder (CYSDV), vein yellowing (SqVYV), chlorotic yellows (CCYV), were used development of OMRT-PCR. gene, rubisco from cucurbits as standardization assay monitor false amplification. In addition, we examined use total nucleic acid template OMRT-PCR infection. demonstrated high sensitivity detected all four infections 1 ng reaction. multiple approach successful diagnosis 16 18 samples collected commercial fields Florida. specific, rapid, sensitive, cost-efficient viruses. This technique expected improve ease CuLCrV, CYSDV, SqVYV CCYV assist monitoring production fields. • simultaneous infecting cucurbits: CCYV. newly specific these limit this up acid. have potential reduce cost, time labor large number samples.