作　　者： ; ; ; ; ;

机构地区： 广东省实验动物监测所

出　　处： 《中国实验动物学报》 2009年第5期354-359,I0007,I0008,共8页

摘　　要： 目的建立小鼠肝炎病毒(mouse hepatitis virus,MHV)逆转录环介导等温扩增(reverse transcription loop-mediated isothermal amplification,RT-LAMP)检测技术并进行初步应用。方法针对MHV核衣壳蛋白基因序列设计6条特异性引物,以MHV基因组RNA为模板,进行RT-LAMP反应。扩增产物通过浊度、SYBR green I荧光染料显色及电泳三种方法观察结果。同时,用限制性内切酶AluI对扩增产物进行酶切鉴定。进行RT-LAMP特异性和敏感性试验。最后用建立的RT-LAMP方法检测55份小鼠临床样本。结果RT-LAMP技术简单快速,等温条件下(65℃)一个小时内即可完成反应。酶切结果证明RT-LAMP产物正确。该技术具有很高的特异性和敏感性,可检测到的MHV RNA最低浓度为0.1 pg/μL,比逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)灵敏10倍。临床样本检测结果显示,与RT-PCR比较RT-LAMP方法的敏感性和特异性分别为100%和83.3%。结论RT-LAMP技术是一种快速、简便诊断方法,该方法检测特异性强、敏感性高,可以成为快速准确检测MHV的有效手段。 Objective To establish a reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for mouse hepatitis virus（MHV）detection,and use this method to test clinical specimens.Methods A set of six specific primers,two outer,two inner and two loop primers were designed based on MHV nucleocapsid protein gene for RT-LAMP assay.Amplification products were detected by visual turbidity,SYBR green I staining and agarose gel electrophoresis.RT-LAMP results were also confirmed by Alu I restriction enzyme digestion.Specificity and sensitivity of RT-LAMP were determined and 52 clinical specimens were tested.Results RT-LAMP procedure was quite simple and rapid,wherein the amplification was carried out under isothermal conditions at 65℃ within 1 h.Digestion with Alu I restriction enzyme demonstrated that the amplified product was unique.It showed a high specificity and the detection limit was 0.1 pg/μL using ten-fold series dilutions of MHV RNA,and RT-LAMP was 10 times more sensitive than reverse transcription-polymerase chain reaction（RT-PCR）.The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100% and 83.3%,respectively.Conclusion The RT-LAMP assay reported here has the advantages of rapid amplification,simple operation,and easy detection as well as high sensitivity and specificity,and may become a useful method for rapid and reliable clinical diagnosis of MHV.

关 键 词： 小鼠肝炎病毒 逆转录环介导等温扩增 逆转录聚合酶链式反应

领　　域： [农业科学] [农业科学] [农业科学] [农业科学] [农业科学]