猪轮状病毒RT-LAMP检测方法的建立及应用

Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine

为了建立一种适用于猪轮状病毒(PoRV)的逆转录-环介导等温核酸扩增(RT-LAMP)的快速、灵敏检测方法。依据GenBank上登录的PoRV VP7基因保守序列,设计了6条特异性引物,通过对外引物与内引物浓度比、Bst DNA聚合酶浓度、Mg2+浓度、dNTP浓度和反应条件等进行优化。结果显示,当外引物与内引物浓度比为200nmol/L∶2 400nmol/L(1∶12)、Bst DNA聚合酶浓度为0.64 U/μL、Mg2+浓度为2.5 mmol/L、dNTP浓度为1.0mmol/L,在恒温(60℃)条件下作用60min,扩增效果出现明显"梯状"条带,同时对建立的RT-LAMP检测方法进行特异性和敏感性验证,其只有PoRV获得特异性扩增条带,与其他猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪瘟病毒等无交叉反应,具有良好的特异性,最低检测分子拷贝数为1.0×102拷贝/μL,具有极高的敏感性。反应结束后肉眼可见阳性扩增产物出现白色沉淀,加入SYBR GreenⅠ观察颜色变化可以判定结果。该方法适用于野外、基层部门和海关快速检测PoRV的新方法,在临床上有良好的推广意义。

We wished to establish a method for rapid and sensitive detection of reverse transcription loopmediated isothermal amplification（RT-LAMP）for the rapid and sensitive detection of porcine rotavirus（PoRV）.According to the published PoRV VP7 sequences in GenBank,6specific primers were designed.According to the concentrations of foward and reverse primers,Bst DNA polymerase,Mg2＋,and dNTP,reaction conditions were optimized.Results revealed the concentration ratio of foward and reverse primers to be 200nmol/L∶2,400nmol（1∶12）,Bst DNA polymerase concentration to be 0.64U/μL,Mg2＋concentration to be 2.5mmol/L,and dNTP concentration to be 1.0mmol/L in 1hat 60℃.The amplification effect achieved a＂ladder＂effect,with amplified bands being shown only for PoRV.RT-LAMP was specific and did not elicit a cross reaction with porcine epidemic diarrhea virus,transmissible gastroenteritis virus of pigs,or classical swine fever virus.The sensitivity of RT-LAMP was 1.0×10^2 copies/μL.After the reaction,inspection by the naked eye revealed positive amplification products to appears as cloudy-white precipitates,and addition of SYBR Green I showed a color change.These data demonstrate that RT-LAMP is suitable for the rapid and sensitive detection of PoRV.