实时荧光PCR快速检测粪便中艰难梭菌方法

Rapid detection of clostridium difficile in human stool by real-time fluorescence PCR

目的:建立实时荧光PCR快速检测艰难梭菌的方法。方法:以艰难梭菌磷酸丙糖异构酶(tpi)基因的保守序列为模板设计和合成特异性引物和荧光标记探针,建立实时荧光PCR检测体系,通过检测含有艰难梭菌标准菌株浓度为106-10 CFU/ml的细菌培养物及加标模拟样本进行敏感性分析,并对其特异性和干扰性进行评价。结果:该方法只对艰难梭菌进行特异性扩增,其他常见的病原菌均不能扩增;整个检测过程只需要2 h,对艰难梭菌菌悬液可检测至10 CFU/ml细菌,对加标粪便样本可检测至1000 CFU/ml细菌。结论:本研究建立的实时荧光PCR检测艰难梭菌方法具有快速、特异、敏感性高等优点,能实现对艰难梭菌的快速检测。

Objective： To develop a real-time fluorescence PCR assay for rapid detection of Clostridium difficile.Methods： The special tpi gene of C.difficile were amplified through designing special primers and TaqMan probes within the conserved and specific regions for this gene.In this way,a rapid and stable method of real-time PCR assay for the detection of C.difficile standard bacterial concentration with 106-10 cfu/ml was established.The specificity and sensitivity of PCR were also analyzed.By adding standard culture fluid in blank fecal sample,the sensitivity and interference of the method was evaluated.Results： The detection limits of pure culture in the real-time PCR assay were 10 CFU/ml.The detection limit for C.difficile in artificially contaminated fecal sample was 103 CFU/ml.Conclusion： These results indicated that the real-time PCR method for C.difficile detection was rapid,high in specificity and sensitivity and suitable for the detection of C.difficile in fecal.