百日咳鲍特菌23S rRNA位点变异AS-PCR检测方法建立

Development and evaluation of a detection method for 23S rRNA mutation of Bordetella pertussis with allele-specific PCR

目的根据allele-specific PCR(AS-PCR)原理,建立具有良好敏感性与特异性的百日咳鲍特菌23S r RNA位点变异检测方法。方法基于百日咳鲍特菌23S r RNA A2047G位点突变与其对红霉素耐药的相关性,设计包含特异变异位点的引物进行两阶段PCR反应,根据电泳有无特异大小目的片段的出现,确定百日咳鲍特菌23S r RNA A2047G位点是否有变异。结果以基因序列测定法为金标准的评价结果显示,AS-PCR法检测突变型菌株的灵敏度为96%(144/150),特异度为100%(100/100),kappa=0.95(P<0.01);检测野生型菌株的灵敏度为100%(18/18),特异度为100%(100/100),kappa=1(P<0.01)。结论 AS-PCR方法检测百日咳鲍特菌23S r RNA A2047G位点变异具有较高灵敏度和特异度,适用于普通微生物实验室开展百日咳鲍特菌对红霉素耐药性的快速检测。

Objective To establish a highly specific and sensitive method to detect 23S rRNA mutation in Bordetella pertussis （B. pertussis） with allele-specific PCR （AS-PCR）. Methods Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, primers containing specific mutation sites were designed to establish the two-stage PCR. High performance capillary electrophoresis was employed to detect the target band to identify the mutation of 23S rRNA. Results Assessed by the golden standard e.g. the results of gene sequencing, the sensitivity is 96% （144/150） and the specificity is 100% （100/100）, with a kappa value of 95 （P 〈 0.01） for the detection of the mutant strains using the established AS-PCR method; while, the sensitivity is 100% （18/18） and the specificity is 100% （100/100）, with a kappa value of 1 （P 〈 0.01） for the detection of wild strains. Conclusion The established AS-PCR method is highly sensitive and specific for the detection of 23S rRNA A2047G mutation in Bordetella pertussis; the method could be applied to rapid detection of rythromycin-resistance of Bordetella pertussis in common clinical microbiology laboratories.