| 李淑华,刘岩,曹广文.利用反转录环介导等温扩增技术检测麻疹病毒[J].Chinese journal of Epidemiology,2014,35(2):186-189 |
| 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 amplification (RT-LAMP) |
| Received:November 21, 2013 |
| DOI：10.3760/cma.j.issn.0254-6450.2014.02.019 |
| KeyWord: 麻疹病毒 反转录环介导等温扩增 反转录－聚合酶链反应 |
| English Key Word: Measles virus Loop-mediated isothermal amplification Reverse transcription-
polymerase chain reaction |
| FundProject:第43批中国博士后科学基金资助（20080431367）；虹口区卫生局课题基金（虹卫1103-33）；上海市自然科学基金（07ZR14141） |
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| Abstract: |
| 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1 h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。 |
| English Abstract: |
| 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℃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 electrophoresis. The appearance of color change in the reactions with positive
of agarose gel samples was evident at 20 min after RT-LAMP initiation. The controls and positive 100-fold higher than that MV specific LAMP assay
sensitivity of RT LAMP assay was of the conventional RT PCR of the real-time RT-PCR. The specificity of 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 |
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