利用反转录环介导等温扩增技术检测麻疹病毒.pdf

《利用反转录环介导等温扩增技术检测麻疹病毒.pdf》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、·186·中华流行病学杂志2014年2月第35卷第2期ChinJEpidemiol，February2014，Vo1．35，No．2·实验室研究·利用反转录环介导等温扩增技术检测麻疹病毒李淑华刘岩曹广文【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60rain，802min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、r

2、ea1timeRT-PCR进行比较。结果RT-LAMP反应约lh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。【关键词】麻疹病毒；反转录环介导等温扩增；反转录一聚合酶链反应Detectionofmeaslesvirusgenomebyreversetranscription—loopmediatedisothermalamplification(RT—LA

3、MP)uhua，LiuYan，CaoGuangwen．1ShanghaiHongkouDistrictCenterforDiseaseControlandPrevention，Shanghai200082，China；2Departmentof印idemiology，CollegeofBasicMedicalSciences，SecondMilitaryMedicalUniversityCorrespondingauthor：CaoGuangwen，Email：gcao@smmu．edu．cnTh／sworkwassuppoaed6vgrantsfromth

4、eForty-thirdPost-doctoralScienceFoundation(No．20080431367)，HongkouDistrictHealthBureau0厂Shanghai(No．1103—33)andNaturalScienceFoundationofShanghai(No．07ZR14141)．【Abstract】0bjectiveToestablishatoolregardingthereversetranscription-loopmediatedisothermalamplification(RT-LAMP)assayforth

5、edetectionofmeaslesvirus．MethodsMeaslesvirusRNAwasextractedbyTrizol-LSreagent．The1242—1442sequencecontained8primersitesof6setsprimer．TheRT．LAMPgeneamplificationwasdetectedbyareal—timePCRfacilitywithAMVreversetranscriptaseat63cCfor60minbeforeterminatingtheamplificationat8O℃2min．Thea

6、mplifiedproductwasmonitoredbyagarosegelelectrophoresisandloopampfluorescencemethods．Sensitivityandspecificityofther_LAMPassayweresubsequentlycomparedwiththatofconventionalRT_PCR．ResultsThewholeproc~：dureofRT-LAMPtookaboutlhour．Theamplifiedproductsappearedtobealadder-likeelectrophor

7、esispattemduringtheprocessofagarosegelelectrophoresis．TheaDpearanceofcolorchangeinthereactionswithpositivecontrolsandpositivesampleswasevidentat20minafterr_LAMPinitiation．ThesensitivityofI己1LAMPassaywas10O—f0ldhigherthanthatoftheconventionalRT-PCRofthereal—timer_PCR．Thespecificityo

8、fMV-specificLAMPassaywasco