环介导等温扩增技术检测蚊体内间日疟原虫子孢子的研究  

Detection of Plasmodium vivax sporozoites-carrying mosquitoes using loop-mediated isothermal amplification (LAMP)

作　　者：朱韩武[1] 曹俊[1] 周华云[1] 李菊林[1] 朱国鼎[1] 顾亚萍[1] 王伟明[1] 刘耀宝[1] 陶志勇[2] 高琪[1] 

机构地区：[1]江苏省寄生虫病防治研究所、卫生部寄生虫病预防与控制技术重点实验室、江苏省寄生虫分子生物学重点实验室、江苏省寄生虫病学重点学科,无锡214064 [2]苏州大学基础医学院寄生虫学教研室

出　　处：《中国血吸虫病防治杂志》2010年第2期158-163,共6页Chinese Journal of Schistosomiasis Control

基　　金：国家重大科技专项经费资助(2008ZX10004-011);第五轮中国全球基金疟疾项目硕士研究生培养专项基金(2008-1);江苏省血地寄科研课题(X200733)

摘　　要：目的建立一种简便、快速和高敏感度的蚊体内间日疟原虫检测的环介导等温扩增方法(LAMP)。方法针对间日疟原虫环子孢子蛋白(CSP)基因种属特异性保守区域6个位点设计2对引物,以感染性按蚊、阴性按蚊、恶性疟原虫及正常人血DNA为模板评价LAMP的特异性。将间日疟原虫CSP基因质粒DNA梯度稀释,并与阴性按蚊DNA按1.0μl加1.3×106、1.3×105、1.3×104、1.3×103、1.3×102、1.3×101、1.3×100拷贝混合后为模板进行LAMP反应,观察其检测敏感性;将感染性按蚊与阴性按蚊DNA作1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256稀释,然后以稀释样本为模板进行LAMP反应,观察批量检测的敏感性。再用此方法与镜检解剖、巢式PCR同时检测同批次人工感染的67只按蚊,评价其应用价值。结果此法检测感染性按蚊阳性,对照组均为阴性;检测不同比例稀释的间日疟原虫CSP基因质粒DNA与按蚊DNA混合物,最低可检测1.3×102拷贝的间日疟原虫CSP基因质粒DNA与按蚊DNA的混合物;检测不同比例感染按蚊与阴性按蚊DNA混合样本,最低可检测出在128个按蚊中有1个感染按蚊的混合样本;用此法检测67只同批次人工感染的按蚊,检出率为47.76%,解剖镜检检出率为25.37%(χ2=7.24,P<0.01),巢式PCR检出率为40.30%(χ2=0.73,P>0.05)。以镜检解剖作为金标准,LAMP敏感性为100%,巢式PCR敏感性为100%。结论LAMP检测蚊体内间日疟原虫简便、快速、敏感性高,具有广阔的应用前景。Objective To establish a simple,convenient,quick and high sensitive method of loop-mediated isothermal amplification （LAMP） for detection of Plasmodium vivax-carrying mosquitoes.Methods The species conservative regions of P.v CSP gene were selected to design 2 pairs of primers which recognized 6 distinct regions.To evaluate the specificity of detection by LAMP,infected Anopheles,An.sinensis （An.s）,Plasmodium falciparum （P.f）,and healthy human blood DNA were selected as templates.To assess the sensitivity of detection,1.3×106,1.3×105,1.3×104,1.3×103,1.3×102,1.3×101 and 1.3×100 copies of P.v CSP plasmid DNA mixed with 1.0 μl An.s DNA were used as the templates of LAMP.The infected An.s DNAs were diluted with negative An.s DNA by 1：2,1：4,1：8,1：16,1：32,1：64,1：128 and 1：256 and then detected by LAMP to show the sensitivity of batch quantity detection.The applied value of this method was evaluated by detecting the same batch of 67 artificial infected An.s mosquitoes,and compared with the detection of microscopic examination and nested PCR in parallel.Results By using LAMP,the detection of infected An.s was positive,while the control samples were all negative.The limits of detection of different proportion dilutions of the mixture of P.v CSP plasmid DNA with An.s DNA were 1.3×102copies.The limits of detection of different proportion dilutions of the mixture of infected An.s DNA with An.s DNA were 1：128.The positive rate of detecting the same batch of 67 artificial infected mosquitoes was 47.76% by LAMP,25.37% by the microscopic examination （χ2=7.24,P0.01）,40.30% by nested PCR（χ2=0.73,P0.05）.Compared with the test of the microscopic examination and then with a statistical analysis,the sensitivity of LAMP was 100%,which agreed well with the sensitivity of nested PCR （100%）.Conclusion The method of LAMP is simple,convenient and high sensitive,and it is a potential method for detecting Plasmodium vivax-carrying mosquitoes in the field.

关 键 词：环介导等温扩增技术 间日疟原虫 子孢子 蚊媒 

分 类 号：R382.31[医药卫生—医学寄生虫学]