中华流行病学杂志
中華流行病學雜誌
중화류행병학잡지
CHINESE JOURNAL OF EPIDEMIOLOGY
2014年
2期
186-189
,共4页
麻疹病毒%反转录环介导等温扩增%反转录-聚合酶链反应
痳疹病毒%反轉錄環介導等溫擴增%反轉錄-聚閤酶鏈反應
마진병독%반전록배개도등온확증%반전록-취합매련반응
Measles virus%Loop-mediated isothermal amplification%Reverse transcription-polymerase chain reaction
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60 min,80℃2 min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-time RT-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-time RT-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.
目的 建立一種快速檢測痳疹病毒的方法.方法 以分離到的痳疹病毒RNA為模闆,利用環介導等溫擴增技術(LAMP)原理,設計閤成3套引物,特異型識彆病毒基因的8箇位點,在反轉錄酶作用下,63℃擴增60 min,80℃2 min終止反應,最終產物分彆經凝膠電泳和熒光目視觀察.通過real-time儀實時鑑測反應過程,同時將該方法的靈敏度、特異度與常規RT-PCR、real-time RT-PCR進行比較.結果 RT-LAMP反應約1h即可完成,最終產物經電泳觀察可見大小片段不等的呈梯度的擴增條帶,目視顯示反應液顏色由黃色變為綠色.靈敏性是常規RT-PCR和real-time RT-PCR的100倍.結論 RT-LAMP方法具有靈敏、特異、快速、簡便、成本低等特點,適用于基層和現場使用.
목적 건립일충쾌속검측마진병독적방법.방법 이분리도적마진병독RNA위모판,이용배개도등온확증기술(LAMP)원리,설계합성3투인물,특이형식별병독기인적8개위점,재반전록매작용하,63℃확증60 min,80℃2 min종지반응,최종산물분별경응효전영화형광목시관찰.통과real-time의실시감측반응과정,동시장해방법적령민도、특이도여상규RT-PCR、real-time RT-PCR진행비교.결과 RT-LAMP반응약1h즉가완성,최종산물경전영관찰가견대소편단불등적정제도적확증조대,목시현시반응액안색유황색변위록색.령민성시상규RT-PCR화real-time RT-PCR적100배.결론 RT-LAMP방법구유령민、특이、쾌속、간편、성본저등특점,괄용우기층화현장사용.
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 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 imerest and proved to be potentially useful for surveillance on MV,especially in the grass root laboratories or for field studies.
