Use of Fluorescent Microsphere-Based Assay for Detection of Three Cucurbit-Infecting Viruses

: In this study, we describe multiplex polymerase chain reaction (PCR) coupled with the LiquiChip assay for the identification of Zucchini yellow mosaic virus, Cucumber green mottle mosaic virus, and Cucumber mosaic virus by coamplification with plant mRNA as an internal control. Multiplex reverse-transcription (RT)-PCR products were subjected to allele-specific primer extension, then hybridized to carboxylated microspheres with unique fluorescent identifiers followed by detection using the LiquiChip 200 workstation. This assay is highly specific for distinguishing individual viruses from a mixed viral population and is 10 times more sensitive than multiplex RT-PCR. In addition, the establishment of this method enabled the detection of cucurbit viruses in field samples.