套式PCR和DNA探针技术检测Q热立克次体

摘要

Aim To establish the rapid and sensitive methods for detection ofCoxiella burnetti (Cb).Methods The nested PCR was established by using two sets of primers designed based on the intervening sequence(IVS) in 23S rRNA gene of Cb and the amplimer of Cb genomic DNA was labeled with 32P as a probe in dot-blot hybridization.Results The positive results were observed in all 9 Cb strains but not in controls in both NPCR and hybridization assays. The nested PCR and IVS probe detected as little as 0.1pg and 0.5ng Cb genomic DNA, respectively. The blood of guinea-pigs, the blood and spleens of mice infected with Cb were positive in both assays. Conclusion The nested PCR and IVS probe are highly sensitive and specific for detection of Cb and they may be more useful in diagnosis of Q fever.%目的 建立套式PCR（NPCR）和DNA探针技术用于检测Q热立克次体。方法 依据已知的Cb23srRNA基因序列，设计两对引物用于建立套式PCR技术，并将扩增片段制成探针进行斑点杂交。结果 9株Cb分离株均可扩增出阳性产物带，而对照菌均为阴性，扩增灵敏度可达0.1pgCbDNA,实验感染第6天的豚鼠血、小鼠血、小鼠脾脏均可扩增出阳性带;用七医株扩增片段制成的探针与9株Cb分离株的全DNA呈阳性杂交，对照菌为阴性，探针杂交灵敏度为0.5ngCbDNA,感染的豚鼠血、小鼠血、小鼠脾脏均可呈阳性反应。结论我们的NPCR和DNA探针技术具有很好的特异性和灵敏度，可将二者联合用于Q热的病原学诊断。