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