炭疽芽孢杆菌防污染PCR-微孔板杂交-EIA检测技术的研究

Anti-contamination PCR-microplate hybridization-enzyme immunoassay for detecting Bacillus anthracis spores

目的 :建立防污染PCR_微孔板杂交_EIA检测技术 ,并用于炭疽芽孢杆菌芽孢的检测。方法 :根据炭疽芽孢杆菌毒力相关基因设计引物 ,筛选合适引物 ,建立用UDG酶防污染的PCR扩增_微孔板杂交与酶联显色检测炭疽芽孢杆菌的方法 ,并探讨试剂的室温稳定系统 ,将建立的方法用于模拟污染炭疽芽孢土壤样品的检测。结果 :根据炭疽芽孢杆菌荚膜和水肿因子基因设计的引物 ,可以同时检测两个毒力相关质粒的存在 ,用 0 .1U的UDG可以防止10 10 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。加入稳定剂的PCR_微孔板杂交_EIA体系可以耐受 7d的 37℃破坏试验。将该体系用于污染土壤的检测 ,可以检测出 0 .2 5g土壤中 10 3 个炭疽芽孢的存在。结论 :所研制的防污染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 。

Objectives:To establish an anti_contamination PCR_microplate hybridization_enzyme immunoassay (EIA) for detecting Bacillus anthracis spores. Methods:The specific primers were designed based on virulence_related genes encoding edema factor in plasmid pXO1 and capsule in pXO2.Uridine glycosylase(UDG) was used in PCR system for digesting the carry_over amplicons and preventing contamination, and the microplate hybridization_EIA was used for verifying the specific amplicons. In order to facilitate the feasibility of this technique,an enzyme stabilizer was introduced into the system to obtain a stable reagent system for convenient application. Results:The primers used in this system could detect two major plasmids in B.anthracis simultaneously. 0.1?U of UDG could digest 10 10 molecules contaminated in the PCR system. The sensitivity of PCR_microplate hybridization was 10 times higher than that demonstrated by PCR_agarose electrophoresis. The stabilizer made the system stable at 37℃ for up to 7 days. The anti_contamination PCR_microplate hybridization_EIA system could detect 10 3 anthrax spores in 0.25?g soil.Conclusions:This technique overcomes the problems in conventional PCR, such as inconvenience for transportation of reagent, false positive due to lontamination and lack of objective evaluation of results by electrophoresis. This is a powerful method for rapid detection of anthrax spores.