防污染PCR-微孔板杂交检测布鲁氏菌方法研究

Anti-contamination PCR-microplate hybridization-enzyme immunoassay for detecting Brucella spp

目的 建立防污染 PCR-微孔板杂交 - EIA检测技术 ,并用于布鲁氏菌及其模拟感染组织中细菌的检测。方法 根据布鲁氏菌 31k D热休克蛋白和外膜蛋白 2 B(OMP2 B)基因设计引物 ,筛选合适引物 ,并建立用脲嘧啶糖基化酶防污染的 PCR扩增 -微孔板杂交与酶联显色检测布鲁氏菌的方法 ,并用于模拟污染布鲁氏菌动物脏器标本的检测。结果 根据布鲁氏菌 31k D热休克蛋白和外膜蛋白 2 B基因设计的引物 ,建立了复合 PCR,同时检测 2个基因的存在 ,并发现不同引物序列对 PCR扩增敏感性影响较大 ,可以相差 10 0 0倍。用 0 .5 U的U DG可以防止 10 8产物的污染 ,微孔板杂交 -酶联显色检测比单纯 PCR-电泳敏感 10倍。将该体系用于污染动物脏器的检测 ,可以检测出反应体系中 2～ 2 0 0个细菌的存在。结论 研究的防污染 PCR-微孔板杂交 - EIA试剂克服了目前 PCR试剂运输不便、产物污染所致假阳性和判定结果欠客观的缺点 。

Objective To establish an anti contamination PCR microplate hybridization enzyme immunoassay (EIA) for detecting Brucella spp.Methods The specific primers were designed based on two genes, encoding 31 KD heat shock protein and outer membrane protein (OMP)2B. UDG was used in PCR system for digesting the carry over amplicons to preventing contamination, and the microplate hybridization EIA used for verifying the specific amplicons.Results A duplex PCR system was developed by using the primers located on two genes of Brucella spp, which was used to detect the two genes simultaneously to prevent the false negative results caused by gene mutation. Our results proved that the primer sequences had a great influences on the sensitivity of PCR. 0.5 U of UDG can digest 10 8 molecules contaminated in the PCR system. The sensitivity showed by PCR microplate hybridization is 10 times higher than that demonstrated by PCR agarose electrophoresis. The anti contamination PCR microplate hybridization EIA system could detect 2 to 200 bacteria in the PCR system.Conclusions This technique overcomes the problems for conventional PCR, that is inconvenience for reagent's transportation, amplicon's carry over and lack of objective evaluation of results by electrophoresis. This is a powerful method for rapid detecting Brucella spp.