Multiplex RT-PCR for simultaneous detection of seven cucurbit viruses found in Florida cucurbits

Virus detection of phytopathogens has benefited greatly from advancements in diagnostic technologies; however, not all laboratories have access to high-cost platforms. To address this limitation, a single-tube multiplex reverse transcription-polymerase chain reaction (m50RT-PCR) assay was developed for the simultaneous detection of six RNA viruses - cucurbit chlorotic yellows virus (CCYV), squash vein yellowing virus (SqVYV), papaya ringspot virus-W (PRSV-W), watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2, cucurbit yellow stunting disorder virus (CYSDV) and one DNA virus, cucurbit leaf crumple virus (CuLCrV), affecting cucurbits in South Florida. The assay detected all seven viruses with a limit of detection of 10 copies per target (theoretical visualization threshold, as detected by BioRad's ImageLab software), with amplicons consistently visualized on an agarose gel at 100 copies (practical visualization threshold, as seen with the naked eye under UV illumination). Specificity was confirmed through sequencing and in silico analyses. Field validation using 65 samples representing diverse hosts, tissues, and collection years (2020-2024) demonstrated diagnostic sensitivity ranging from 92 to 100%, specificity from 95 to 100%, and overall accuracy from 95 to 100%. A limitation of the assay is cross-reactivity between CCYV and CYSDV, likely due to sequence similarity within the genus Crinivirus. To address this, a secondary multiplex RT-PCR assay was developed to differentiate these viruses when required. Despite this additional step for a subset of samples, the m50RT-PCR assay reduces cost and turnaround time compared to individual assays and provides an accessible tool for cucurbit virus diagnostics and surveillance.