Development of simplex and quintuplex RT-PCR for simultaneous detection of soybean viruses

IF 2.2 4区 医学 Q3 BIOCHEMICAL RESEARCH METHODS Journal of virological methods Pub Date : 2024-08-31 DOI:10.1016/j.jviromet.2024.115010
K. Kalaiponmani , B. Parameswari , A. Tripathi , V. Celia Chalam
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Abstract

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 10−4 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 10−3 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.

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开发用于同时检测大豆病毒的简单和五重 RT-PCR 技术。
为检测和区分感染大豆的豆荚斑驳病毒 (BPMV)、樱桃卷叶病毒 (CLRV)、覆盆子环斑病毒 (RpRSV)、大豆花叶病毒 (SMV) 和番茄环斑病毒 (ToRSV),开发了五种单体和一种多重 RT-PCR (m-RT-PCR) 方案。简易 RT-PCR 方案产生的病毒特异性扩增子分别为:BPMV 538bp、CLRV 139bp、RpRSV 298bp、SMV 403bp 和 ToRSV 282bp,灵敏度低至 10-4 稀释 cDNA。此外,为了在单管中同时检测所有五种病毒,还优化了五重 RT-PCR 方案,稀释的 cDNA 低至 10-3,引物为 0.05µM。为了验证单工 RT-PCR 方案的可靠性,对进口大豆样品进行了 ELISA 和 RT-PCR 检测。结果表明,所开发的方案可以检测进口大豆中的病毒,而且在解决种传病毒检测的模糊性方面比 ELISA 更有效。所开发的单复性和五复性 RT-PCR 方案将有助于在检疫处理过程中诊断大豆种质是否合并感染了病毒,以确保国家基因库中的种子长期无病毒保存,并有助于检疫认证。
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来源期刊
CiteScore
5.80
自引率
0.00%
发文量
209
审稿时长
41 days
期刊介绍: The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. The methods may include, but not limited to, the study of: Viral components and morphology- Virus isolation, propagation and development of viral vectors- Viral pathogenesis, oncogenesis, vaccines and antivirals- Virus replication, host-pathogen interactions and responses- Virus transmission, prevention, control and treatment- Viral metagenomics and virome- Virus ecology, adaption and evolution- Applied virology such as nanotechnology- Viral diagnosis with novelty and comprehensive evaluation. We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.
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