罗氏沼虾野田村病毒实时荧光定量RT-PCR检测方法的建立  

Establishment of SYBR Green I real-time RT-PCR method for detecting Macrobrachium rosenbergii nodavirus

作　　者：黄光华 童桂香 黎小正 李旻 黄国秋 卢小花 陈静 吴明媛 韦信贤 

机构地区：[1]广西水产科学研究院,南宁530021

出　　处：《南方农业学报》2015年第11期2059-2063,共5页Journal of Southern Agriculture

基　　金：广西水产畜牧兽医局科技推广应用项目(桂渔牧科201452005;桂渔牧科201452037);广西直属公益性科研院所基本科研业务费项目(GXIF-2012-08)

摘　　要：【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,Tm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Ct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]The present experiment was conducted to establish SYBR Green I real-time RT-PCR method for detecting Macrobrachium rosenbergii nodavirus（MrNV）,in order to provide technical support for prevention and control of white tail disease（WTD）.[Method]The gene fragment（No.HQ287005） of MrNV capsid protein were cloned into pGM-T vector.And the positive linearized plasmids,which were obtained through restriction enzyme Sal I digestion,was used as template DNA for transcription.The RNA standards were obtained through in vitro transcription,so as to make standard curve.A pair of specific primers was designed and synthesized to amplify 119 bp gene fragments based on conserved domains of MrNV,and the concentration of primer was optimized by using RNA standards as template.Then the standard curve and melting curve obtained by using established method were analyzed,and the specificity,sensitivity and repeatability of method were determined.Furthermore,the clinical samples were detected by using established method.[Result]The results showed that,the established one-step SYBR Green I real-time RT-PCR method had advantage of wide quantitative range,and the standard curve presented good linear relation when the detection template ranged from 1×10~1 to 1×10~8 copies/μL.Besides,the melting curve had single crest,and T_m was 81.17-81.25 ℃.The amplification plot presented S-shaped curve,when the cycle threshold（C,） was defined as 35.00,the established method had a detection limit of 10 MrNV copies,which was 1000 times sensitivity of routine RT-PCR.In addition,the established method had high specificity to detect MrNV.The variation coefficient of C,values for intra-assay and inter-assay were 0.15%-0.83%and 0.56%-0.95%,respectively.Furthermore,83 clinical samples were detected by one-step SYBR Green I real-time RT-PCR,and the detection results had no differences from results obtained by nested RT-PCR method.[Conclusion]The established one-step SYBR Green I real-time RT-PCR method is fast,sensitive,accurate,re

关 键 词：罗氏沼虾 野田村病毒 白尾病 一步法实时荧光定量RT-PCR 检测 

分 类 号：S945.41[农业科学—水产养殖]