实时荧光定量PCR检测人粪便中空肠弯曲杆菌方法的建立与评价

Establishment and Application of a Real-Time Quantitative PCR Assay for Detection of Campylobacter Jejuni in Human Feces

目的：建立一种快速检测人粪便样本中空肠弯曲杆菌的实时荧光定量 PCR方法。方法根据空肠弯曲杆菌保守序列设计特异引物，建立空肠弯曲杆菌的实时荧光定量 PCR检测法。对该方法的线性范围、抗干扰能力进行考察。同时采用培养法和实时荧光定量 PCR法对150例粪便样本进行检测，以培养法检测结果作为参照标准，对实时荧光定量 PCR方法的灵敏度、特异度、准确度和重复性进行考察。采用 Kappa检验对两种检测方法的结果进行统计学分析。结果建立的实时荧光定量PCR方法标准曲线线性关系良好Y＝-3.51 Log（X）＋37.09，相关系数为0.996，理论检测下限为102 CFU/ml。抗干扰能力强，仅空肠弯曲杆菌出现特异性扩增曲线。与培养法相比，其灵敏度、特异度、准确度分别为92.4％，95.8％和94％；检测结果重复性良好（CV％＜5％）。统计学分析显示两种方法检测结果具有一致性，且一致性强度为极强（Kappa＝0.88，P＜0.05）。结论实时荧光定量PCR法能准确快速检测粪便样品中的空肠弯曲杆菌。

Objective To establish a real-time quantitative PCR assay for the identification of Campylobacter jejuni in fecal samples.Methods Specific primers of the PCR were designed according to the conserved sequences of Campylobacterjeju-ni,and the real-time quantitative PCR assay was established.150 cases of fecal samples were tested by both culture and PCR methods.With the culture testing results as the reference standard,the sensitivity,specificity,accuracy and repetition of the real-time quantitative PCR were validated.Kappa test was used to estimate the difference between the two detection meth-ods.Results The standard carve of the real-time quantitative PCR assay fitted the equationY=-3.51Log（X）＋37.09 （R2=0.996）well.The sensitivity,specificity,and accuracy of the established method were 92.4%,95.8% and 94%,respective-ly.The theoretical detection limit of the PCR method was 102 CFU/ml,and its reproducibility was good （CV〈5%）.Statisti-cal analysis demonstrated that the results of the two methods were consistent,and the consistent strength was very strong （Kappa=0.88,P〈0.05）.Conclusion The established real-time PCR method can assay the Campylobacterjejuni in human fecal samples rapidly and accurately.