Division of Plant Quarantine, ICAR-NBPGR, New Delhi, India.
ICAR-NBPGR Regional Station, Hyderabad, India.
J Virol Methods. 2024 Dec;330:115010. doi: 10.1016/j.jviromet.2024.115010. Epub 2024 Aug 31.
Five simplex and a multiplex-RT-PCR (m-RT-PCR) protocols were developed for detection and differentiation of bean pod mottle virus (BPMV), cherry leaf roll virus (CLRV), raspberry ringspot virus (RpRSV), soybean mosaic virus (SMV) and tomato ringspot virus (ToRSV) infecting soybean. The simplex RT-PCR protocols produced virus-specific amplicons of 538 bp for BPMV, 139 bp for CLRV, 298 bp for RpRSV, 403 bp for SMV, and 282 bp for ToRSV, with sensitivity down to 10 diluted cDNA. Further, to detect all the five viruses simultaneously in a single tube a quintuplex RT-PCR protocol was optimized with as low as 10 diluted cDNA and 0.05 µM primer. To validate the reliability of the simplex RT-PCR protocol, imported soybean samples were tested by ELISA as well as RT-PCR. The results revealed that the developed protocol could detect the viruses in imported soybean, and found to be efficient than ELISA in resolving ambiguity in detection of seed borne viruses. The developed simplex and quintuplex RT-PCR protocol will be quite helpful for the diagnosis of soybean germplasm co-infected with viruses during the quarantine processing for ensuring virus free long term seed conservation in the National Gene Bank as well as for quarantine certification.
针对侵染大豆的菜豆荚斑驳病毒（BPMV）、樱桃皱叶病毒（CLRV）、黑莓潜隐环斑病毒（RpRSV）、大豆花叶病毒（SMV）和番茄环斑病毒（ToRSV），开发了 5 个单重 RT-PCR （simplex RT-PCR）和 1 个五重 RT-PCR （multiplex-RT-PCR）协议，用于检测和区分上述病毒。单重 RT-PCR 协议能特异性扩增出 538bp 的 BPMV、139bp 的 CLRV、298bp 的 RpRSV、403bp 的 SMV 和 282bp 的 ToRSV 产物，检测灵敏度低至 10 倍稀释的 cDNA。此外，为了在单个管中同时检测所有 5 种病毒，优化了五重 RT-PCR 协议，其检测灵敏度低至 10 倍稀释的 cDNA 和 0.05μM 的引物。为了验证单重 RT-PCR 协议的可靠性，用 ELISA 和 RT-PCR 对进口大豆样品进行了测试。结果表明，该开发的协议能够检测到进口大豆中的病毒，并且在解决种子携带病毒检测中的歧义方面比 ELISA 更有效。开发的单重和五重 RT-PCR 协议将有助于在检疫处理期间对同时感染病毒的大豆种质进行诊断，以确保在国家基因库中进行长期无病毒种子保存以及进行检疫认证。
