摘要
目的 建立防止产物污染的UDPE(UDG -DuplexPCR -EIA)技术 ,用于检测类鼻疽伯克霍尔德氏菌。方法 选择类鼻疽伯克霍尔德氏菌FUR(FerricUptakeRegulator)基因为靶序列 ,设计两对引物进行PCR扩增 ,用UDG(尿嘧啶糖基化酶 )防止PCR产物污染 ,并用微孔板杂交 -酶联显色检测扩增的PCR产物片段 ;用不同的模板考察方法的特异性和灵敏度。结果 该检测系统可以特异性地检测类鼻疽伯克霍尔德氏菌 ;对于纯DNA模板 ,检测灵敏度可以达到 10fg/ μl;对于系列稀释菌液提取的模板 ,灵敏度可达 0 1个菌 / μl;对于模拟污水标本、模拟组织标本和模拟土壤标本的检测灵敏度分别为 10个菌 / μl,10 0个菌 / μl和 10 0个菌 / μl;检测系统可以防止 10 9个PCR产物分子的污染 ;检测系统可以在 37℃下稳定保存 7d。结论 本研究建立了稳定的 ,防止产物污染 。
Aim To develop and objective,rapid,specific and sensitive UDPE (UDG-Duplex PCR-EIA)assay to detect the pathogen Burkholderia pseudomallei.Method Two sets of primers,targeting FUR(Ferric Uptake Regulator)gene of Burkholderia pseudomallei,were chosen to amplify certain fragments,and an enzyme called UDG(Uracil DNA Glycosylase)was added into the PCR reaction mixture to avoid carry-over caused by PCR products.The PCR products were detected by microwell-hybriadation and Enzyme-Immunoassay(EIA).Different templates were employed to evaluate the specificity and sensitivity of this detection system.Results This system can specifically detect Burkholderia pseudomallei without false positive result in realted species.0.1U of UDG in the PCR reaction mixture can effectively prevent carry-over raised by 109 PCR product molecules.The detection limit of pure DNA template is as low as 10fg/μl;when applied to serial dilution bacteria suspension,the detection limit can be 0.1CFU/μl;When applied to ortificial water Somple,the detection limit can be 10 LFU/μl and the sensitivity of detecting artificial tissue sample and soil sample can be 100CFU/μl.The system developed here can maintain stable at 37℃ fo 7 days.Conclusion The UDPE assay developed in this study can serve as a sensitive and specific method to detect Burkholderia pseudomallei,free from the carry-over of PCR products.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2001年第2期35-37,共3页
Chinese Journal of Zoonoses
