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

JARQ : Japan Agricultural Research Quarterly
ISSN 00213551
書誌レコードID(総合目録DB) AA0068709X
本文フルテキスト
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.
刊行年月日
作成者 Shuhei ADACHI-FUKUNAGA Yasuhiro TOMITAKA
著者キーワード Cucumis sativus Cucurbitaceae diagnosis touch-down RT-PCR
公開者 Japan International Research Center for Agricultural Sciences
受付日 2023-02-21
受理日 2024-06-10
オンライン掲載日
59
1
開始ページ 27
終了ページ 38
DOI 10690/jarq.59.27
言語 eng

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