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【目的】建立一种检测罗氏沼虾野田村病毒(Macrobrachium rosenbergii nodavirus,MrNV)的实时荧光定量RT-PCR,为防控罗氏沼虾白尾病(White tail disease,WTD)提供技术支持。【方法】将MrNV衣壳蛋白基因片段(No.HQ287005)克隆到pGM-T载体,选取阳性重组质粒用SalⅠ酶切获得线性化转录模板DNA,经体外转录获得标准品RNA;在MrNV基因序列的保守区域设计1对扩增片段为119 bp的特异性引物,以标准品RNA为模板进行引物浓度筛选及标准曲线制定,并对建立的检测方法进行标准曲线和融解曲线分析及特异性、敏感性、重复性、临床样品检验试验。【结果】建立的一步法实时荧光定量RT-PCR定量范围宽,检测模板范围在1×10~1~1×10~8拷贝/μL时,其标准曲线呈良好的线性关系;融解曲线表现为单一波峰,T_m为81.17~81.25℃;扩增曲线呈明显的S形,以C_t值35.00为界限,灵敏度约为10个病毒粒子,是常规RT-PCR的1000倍。该方法仅对MrNV具有良好的特异性,其组内和组间试验的C_t值变异系数分别为0.15%~0.83%和0.56%~0.95%;对83份罗氏沼虾临床样品进行检测,其结果与套式RT-PCR的检测结果一致。【结论】针对MrNV检测建立的一步法实时荧光定量RT-PCR具有快速、便捷、灵敏、准确等优点,且对样品的检测范围宽,适用于MrNV隐性感染和发病诊断。
【Objective】 To establish a real-time fluorescence quantitative RT-PCR for detecting Macrobrachium rosenbergii nodavirus (MrNV) and provide technical support for the prevention and control of White tail disease (WTD). 【Method】 The gene fragment of MrNV capsid protein (No.HQ287005) was cloned into pGM-T vector and the positive recombinant plasmid was selected to obtain the linearized transcription template DNA by Sal Ⅰ digestion, and the standard RNA was obtained by in vitro transcription. In the sequence of MrNV gene Conserved region design A pair of primers with a specific fragment of 119 bp was amplified with standard RNA as a template and the standard curve was established. The standard and melting curve analysis and specificity and sensitivity , Repeatability, clinical sample test. 【Result】 The established one-step real-time quantitative RT-PCR showed a wide quantitative range. The calibration curve showed a good linearity when the detection range was 1 × 10 ~ 1 ~ 1 × 10 ~ 8 copies / μL. A single peak, T_m 81.17 ~ 81.25 ℃; amplification curve was significantly S-shaped C_t value of 35.00 as the limit, the sensitivity of about 10 virions, 1000 times the conventional RT-PCR. The method only has good specificity for MrNV, and the coefficient of variation of C_t value in intra-group and inter-group were 0.15% -0.83% and 0.56% -0.95% respectively. The clinical samples of 83 Macrobrachium rosenbergii samples were detected, and the results It is consistent with the results of nested RT-PCR. 【Conclusion】 The one-step real-time fluorescence quantitative RT-PCR for MrNV detection has the advantages of fast, convenient, sensitive and accurate, and has a wide range of detection for MrNV latent infection and diagnosis.