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].中华流行病学杂志,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) |
收稿日期:2013-11-21 出版日期:2014-09-17 |
DOI:10.3760/cma.j.issn.0254-6450.2014.02.019 |
中文关键词: 麻疹病毒 反转录环介导等温扩增 反转录-聚合酶链反应 |
英文关键词: Measles virus Loop-mediated isothermal amplification Reverse transcription-
polymerase chain reaction |
基金项目: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) |
|
摘要点击次数: 3036 |
全文下载次数: 1102 |
中文摘要: |
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。 |
英文摘要: |
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 |
查看全文
Html全文
查看/发表评论 下载PDF阅读器 |
|
关闭 |