Instituto Valenciano de Investigaciones Agrarias, 46113 Moncada, Valencia, Spain.
J Virol Methods. 2011 Sep;176(1-2):32-7. doi: 10.1016/j.jviromet.2011.05.027. Epub 2011 May 27.
A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.
建立了一种使用通用引物和三种 TaqMan(®)MGB 探针的定量实时逆转录聚合酶链反应（RT-qPCR）程序，用于番茄斑萎病毒（TSWV）基因组 M 片段的通用和基因型特异性检测和定量。使用与三种探针同源的 RNA 转录本制作标准曲线，可以进行重复性好、动态范围宽（10(3)-10(10) TSWV M 片段 RNA 拷贝/ng 总 RNA）且灵敏度高的定量检测。该方案在三种植物宿主的单种或混合感染中，以及在蓟马传播媒介西方花蓟马中，对一系列涵盖目前已知遗传变异范围的 TSWV 分离株进行了检测。这种定量检测方法将成为分子生物学和流行病学研究、诊断和疾病控制的有用工具。
