利用反转录环介导等温扩增技术检测麻疹病毒

Detection of measles virus genome by reverse transcription-loop mediated isothermal amplification （RT-LAMP）

目的建立一种快速检测麻疹病毒的方法。方法以分离到的麻疹病毒RNA为模板，利用环介导等温扩增技术（LAMP）原理，设计合成3套引物，特异型识别病毒基因的8个位点，在反转录酶作用下，63℃扩增60min，80℃2min终止反应，最终产物分别经凝胶电泳和荧光目视观察。通过real—time仪实时监测反应过程，同时将该方法的灵敏度、特异度与常规RT-PCR、real-timeRT-PCR进行比较。结果RT-LAMP反应约1h即可完成，最终产物经电泳观察可见大小片段不等的呈梯度的扩增条带，目视显示反应液颜色由黄色变为绿色。灵敏性是常规RT-PCR和real—timeRT-PCR的100倍。结论RT-LAMP方法具有灵敏、特异、快速、简便、成本低等特点，适用于基层和现场使用。

Objective To establish a tool regarding the reverse transcription-loop mediated isothermal amplification （RT-LAMP） assay for the detection of measles virus. Methods Measles virus RNA was extracted by Trizol-LS reagent. The 1 242-1 442 sequence contained 8 primer sites of 6 sets primer. The RT-LAMP gene amplification was detected by a real-time PCR facility with AMV reverse transcriptase at 63 % for 60 min before terminating the amplification at 80 ~C 2 min. The amplified product was monitored by agarose gel electrophoresis and loop amp fluorescence methods. Sensitivity and specificity of the RT-LAMP assay were subsequently compared with that of conventional RT-PCR. Results The whole procedure of RT-LAMP took about 1 hour. The amplified products appeared to be a ladder-like electrophoresis pattern during the process of agarose gel electrophoresis. The appearance of color change in the reactions with positive controls and positive samples was evident at 20 min after RT-LAMP initiation. The sensitivity of RT-LAMP assay was 100-fold higher than that of the conventional RT-PCR of the real-time RT-PCR. The specificity of MV-specific LAMP assay was conformed by negative amplification of dengue virus and Japanese encephalitis virus. Conclusion RT-LAMP assay appeared rapid, cost-effective, highly sensitive and specific for the detection of genes of interest and proved to be potentially useful for surveillance on MV, especially in the grass root laboratories or for field studies.