A Multiplex RT-PCR Assay for the Detection of Nine Virus Species Infecting Cucumber in Japan

A multiplex one-step reverse transcription polymerase chain reaction (multiplex RT-PCR) assay was developed to detect nine important viruses that infect cucumber in Japan: beet pseudoyellows virus watermelon mosaic virus (WMV), watermelon silver mottle virus (WSMoV), and zucchini yellow mosaic virus (ZYMV). We newly designed virus species-specific primer pairs for seven viruses and used previously reported primer pairs for two viruses. Specificity and sensitivity tests by simplex RTPCR using the primer set showed that touch-down RT-PCR effectively reduced non-specific amplification and had high sensitivity (10 - 10(5)-fold dilution). For multiplex RT-PCR, the primer set was first divided into two sets: primer set I was for WMV, CCYV, KGMMV, BPYV, and WSMoV; primer set II was for PRSV, ZYMV, CMV, and MYSV. After optimizing the ratio of nine primer pairs and the number of cycles of the multiplex RT-PCR, all viruses could be detected by the same PCR condition. When the assays were applied to cucumber samples obtained from an open field and greenhouses, viral infections were clearly identified without non-specific amplification. Therefore, the multiplex RT-PCR assay can be used for the routine diagnosis of the nine viruses in field-growing samples.