Simultaneous detection of three common potyviruses infecting cucurbits by multiplex reverse transcription polymerase chain reaction assay

Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ring spot virus (PRSV-W) are the three most common potyviruses infecting cucurbits in the United States of America. In this study, a Multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection and differentiation of WMV, ZYMV, and PRSV-W. A mixture of specific primers set for each virus successfully amplified a distinct PCR product of 980 bp from the coat protein (CP) gene of WMV, 708 bp from the cylindrical inclusion (CI) gene of ZYMV and 496 bp from the helper component (tic-Pro) gene of PRSV-W. Sanger sequencing confirmed that the amplified PCR products for each virus are specific. The sensitivity and specificity of these primers were tested by serial dilution assay of total RNA extracted from virus-infected samples for all three viruses. The detection limit of the Multiplex RT-PCR assay was between 10(-5)-10(-6)-fold dilution. The Multiplex RT-PCR assay was successfully applied for the detection of these viruses on 54 field samples, which were collected from two counties of Oklahoma during the 2018 growing season. Based on these results, this Multiplex RT-PCR assay is specific, rapid, and economical for the detection of three common potyviruses of cucurbits and has the potential to screen a large number of field samples of cucurbits against these viruses.