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

Japan Agricultural Research Quarterly
ISSN 00213551
NII recode ID (NCID) AA0068709X
Full text
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 (BPYV), cucurbit chlorotic yellows virus (CCYV), cucumber mosaic virus (CMV), kyuri green mottle mosaic virus (KGMMV), melon yellow spot virus (MYSV), papaya ringspot virus (PRSV), 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 RT-PCR using the primer set showed that touch-down RT-PCR effectively reduced non-specific amplification and had high sensitivity (10 - 105-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.
Date of issued
Creator Shuhei ADACHI-FUKUNAGA Yasuhiro TOMITAKA
Subject Cucumis sativus Cucurbitaceae diagnosis touch-down RT-PCR
Publisher Japan International Research Center for Agricultural Sciences
Received Date 2023-02-21
Accepted Date 2024-06-10
Available Online
Volume 59
Issue 1
spage 27
epage 38
DOI 10690/jarq.59.27
Language eng

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