半套式PCR和DNA探针技术检测Q热立克次体

目的 建立半套式PCR和DNA探针技术由于检测Q热立克次体。方法 依据已知的Q热立克次体的16S和23S rRNA基因及其间区的序列。设计3条引物，建立了扩增16S－23S mRNA基因间区的半套式PCR方法，并将扩增片段制成探针，建立了斑点杂交技术。结果 10株Q热立克次体分离株均可扩增出572bp的PCR条带，而对照菌都为阴性，此半套式PCR方法的灵敏度高达1pg；用九里株扩增片段标记后制成的探针，与10个Q热立克次体分离株的全DNA呈阳性杂交，对照菌为阴性，此杂交方法的灵敏度为0．5ng。结论 基于Q热立克次体16S－23S rRNA基因间区的半套式PCR和DNA探针技术具有较好的特异性和灵敏度，可联合应用于Q热的病原学诊断。

Detection of Coxiella burnetii by semi-nested PCR and DNA probe

Objective To establish a rapid and sensitive method for detection of Coxiella buretii . Methods The semi nested PCR had been established by using 3 primers designed based on the sequence of the 16S rRNA and 23S rRNA genes and their intergenic region and the amplimer of Coxiella burnetii genomic DNA was labeled with P32 as a probe in dot blot hybridization. Results The predicted products were amplified from the genomic DNA of all 10 Coxiella burnetii isolates but not from controls in both semi nested PCR and hybridization assays. The semi nested PCR and probe detected as little as 1pg and 0.5ng of Coxiella burnetii genomic DNA,respectively. Conclusion The semi nested PCR and DNA probe are highly sensitive and specific for detection of Coxiella burnetii and can be widely used in the diagnosis of Q fever.