一步法 RT-PCR 同时检测和鉴定四种甘蔗病毒。
Simultaneous detection and identification of four sugarcane viruses by one-step RT-PCR.
机构信息
Laboratory of Plant Virology, South China Agricultural University, Guangzhou 510642, China.
出版信息
J Virol Methods. 2009 Dec;162(1-2):64-8. doi: 10.1016/j.jviromet.2009.07.015. Epub 2009 Jul 29.
Sugarcane mosaic disease (SMD) caused by the Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV) and sugarcane yellow leaf disease (SYLD) caused by the Sugarcane yellow leaf virus (SCYLV) are the two most prevalent and economically important viral diseases of sugarcane. In this study, a one-step quadruplex reverse transcription (RT)-PCR method that employed virus-specific primers was developed for the simultaneous detection and differentiation of SCMV, SrMV, SCSMV and SCYLV. Several sets of primers for each target virus were evaluated for their sensitivity and specificity by simplex and quadruplex RT-PCR. The optimum primer combinations and concentrations, RT temperature and time, and PCR annealing temperature and extension time were determined for the quadruplex RT-PCR. The assay was then validated using sugarcane samples affected with SMD and/or SYLD collected from sugarcane breeding fields and farmers' fields in southern China.
甘蔗花叶病（SMD）由甘蔗花叶病毒（SCMV）、高粱花叶病毒（SrMV）和甘蔗条纹花叶病毒（SCSMV）引起，甘蔗黄叶病（SYLD）由甘蔗黄叶病毒（SCYLV）引起，是两种最普遍和经济上最重要的甘蔗病毒病。在本研究中，开发了一种一步四重反转录（RT）-PCR 方法，该方法使用病毒特异性引物，可同时检测和区分 SCMV、SrMV、SCSMV 和 SCYLV。通过单重和四重 RT-PCR 评估了针对每个靶病毒的多套引物的灵敏度和特异性。确定了四重 RT-PCR 的最佳引物组合和浓度、RT 温度和时间以及 PCR 退火温度和延伸时间。然后，使用从中国南方甘蔗育种田和农民田采集的受 SMD 和/或 SYLD 影响的甘蔗样品对该检测方法进行了验证。
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