Zhang Xun, Wang Xifeng, Zhou Guanghe
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2, West Yuan Ming Yuan Road, Beijing 100094, China.
J Virol Methods. 2008 Aug;151(2):181-187. doi: 10.1016/j.jviromet.2008.05.024. Epub 2008 Jun 30.
Rice stripe virus (RSV) is an important pathogen affecting rice production in subtropical and temperate regions. One-step real time RT-PCR methods using the TaqMan probe are described for quantitative detection of RSV in rice tissues and in Laodelphax striatellus Fallen, the small brown planthopper (SBPH). Primers and probe for specific detection of RSV were designed within the conserved region identified within the coat protein (CP) gene sequence. A DNA fragment was amplified for mimicking the complementary RNA by PCR-based gene assembly, and was used for generation of standard RNA templates. A sensitivity assay showed that the detection limit of the assay was 20 copies, and the standard curve had a linear range from 20 to 2 x 10(5) copies, with good reproducibility. An internal control assay designed to target the rice ubiquitin 5 gene (UBQ5) was included in detecting RSV in different infected rice tissues. Simultaneously, a real time RT-PCR assay was used to detect the RSV CP gene in viruliferous SBPH. The results showed that the numbers of RSV CP genes in different tissues were variable. Accumulation of the RSV CP gene was greater in rice leaf and SBPH thoraco-abdominal tissue than in rice stem and SBPH head, respectively. As determined by an end-point dilution comparison, one-step real time RT-PCR was close to 10(4)-fold more sensitive than the indirect enzyme-linked immunosorbent assay (ELISA) for RSV detection. This quantitative detection assay provides a valuable tool for diagnosis and molecular studies of RSV biology.
水稻条纹病毒（RSV）是影响亚热带和温带地区水稻生产的一种重要病原体。本文描述了使用TaqMan探针的一步法实时RT-PCR方法，用于定量检测水稻组织和灰飞虱（Laodelphax striatellus Fallen）中的RSV。在外壳蛋白（CP）基因序列中鉴定出的保守区域内设计了用于特异性检测RSV的引物和探针。通过基于PCR的基因组装扩增出一个DNA片段以模拟互补RNA，并将其用于生成标准RNA模板。灵敏度分析表明，该检测方法的检测限为20个拷贝，标准曲线的线性范围为20至2×10⁵个拷贝，具有良好的重复性。在检测不同感染水稻组织中的RSV时，纳入了针对水稻泛素5基因（UBQ5）的内对照检测。同时，使用实时RT-PCR检测法检测带毒灰飞虱中的RSV CP基因。结果表明，不同组织中RSV CP基因的数量各不相同。RSV CP基因在水稻叶片和灰飞虱胸腹组织中的积累分别比在水稻茎和灰飞虱头部中的积累更多。通过终点稀释比较确定，一步法实时RT-PCR检测RSV的灵敏度比间接酶联免疫吸附测定（ELISA）高近10⁴倍。这种定量检测方法为RSV生物学的诊断和分子研究提供了一种有价值的工具。
