PCR及PCR-ELISA法检测蚊体内马来丝虫幼虫  

DETECTION OF BRUGIA MALAYI LARVA IN MOSQUITO VECTOR BY PCR AND PCR ELISA *

作　　者：刘云光[1] 孙德建[1] 薛海筹[1] 

机构地区：[1]中国预防医学科学院寄生虫病研究所

出　　处：《中国寄生虫学与寄生虫病杂志》1998年第4期274-278,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：世界卫生组织热带病研究与培训专门规划司资助

摘　　要：目的：建立一种检测蚊体内马来丝虫幼虫的灵敏、快速、特异的方法。方法：选择应用ＰＣＲ及ＰＣＲ－ＥＬＩＳＡ法检测马来丝虫幼虫ＤＮＡ的最佳反应条件，并在该条件下分别以马来丝虫Ⅰ期、Ⅱ期、Ⅲ期幼虫各１条作模板，测定检测的灵敏度及检测实验室人工感染中华按蚊体内的马来丝虫幼虫。结果：ＰＣＲ及ＰＣＲ－ＥＬＩＳＡ法均能检测出１条Ⅰ期幼虫（Ｌ１），而ＰＣＲ的检测下限为１／１０条Ｌ１，ＰＣＲ－ＥＬＩＳＡ检测下限为１／１００条Ｌ１；将分离的感染期幼虫加阴性蚊媒进行粗提、扩增及电泳，结果未见明显的扩增条带，扩增产物作ＥＬＩＳＡ检测，全部为阴性；个体解剖人工感染的中华按蚊１２０只，分别收集１１３只阳性蚊体内的幼丝虫，用两种方法检测，结果均为阳性。结论：初步建立了ＰＣＲ及ＰＣＲ－ＥＬＩＳＡ法检测蚊体内马来丝虫幼虫的方法。AIM: To develop sensitive, specific, simple assays for the detection of Brugia malayi larva in mosquito vectors. METHODS: The optimum conditions for PCR and PCR ELISA were determined. With dissected larvae, detection limits and as well as specificities of the two assays were worked out. The optimized assays were then tested with infected and non infected mosquito vectors, Anopheles sinensis. RESULTS: Both PCR and PCR ELISA detected DNA equivalent to a single L1 stage larva(L1). While the actual detecting limits of the two, as estimated and titrated respectively, reached to 1/10 and 1/100 of a L1. When the two assays were used to detect the larvae isolated from 113 infected A.sinensis, all gave specific bands and positive reactions.However, trials on direct amplifications with crude extracts of infected mosquito vectors consistently failed to give specific bands upon electrophoresis and negative results in PCR ELISA as well. CONCLUSION: Both PCR and PCR ELISA were preliminarily established for the detection of isolated B.malayi larva in mosquito vector , which proved to be sensitive, specific and easy to manipulate.

关 键 词：马来丝虫 聚合酶链反应 ELISA 幼虫 蚊媒 

分 类 号：R383.16[医药卫生—医学寄生虫学] R384.1[医药卫生—基础医学]