三重TaqMan qPCR检测呼吸道感染衣原体方法的建立及应用

Establishment and application of triple TaqMan probe qPCR assay for detection of Chlamydia in respiratory tract

目的本试验的目的是建立可以快速鉴别诊断呼吸道感染肺炎衣原体(Cpn)、鹦鹉热衣原体(Cps)和沙眼衣原体(Ct)的三重TaqMan qPCR方法,有助于衣原体病的科学防控和指导临床用药,为公共卫生安全提供保障。方法选择Cpn、Cps和Ct特异基因ARGR、ompA和ORF8设计引物,建立标准曲线,优化反应条件和体系,验证方法的灵敏度、重复性;通过单独检测3种衣原体的荧光定量PCR方法验证本研究建立方法的准确性。结果结果显示,所建立的三重TaqMan qPCR方法特异度和重复性好,检测灵敏度分别为45 copy/μL(Cpn)、40 copy/μL(Cps)和43 copy/μL(Ct);利用该方法对600个临床样本进行检测,其检出率与参考方法比较,差异无统计学意义(P>0.05),表明该方法检测结果可靠。结论本研究建立的检测呼吸道感染衣原体的三重TaqMan qPCR方法特异性和重复性好,灵敏度高,结果可靠,为呼吸道感染衣原体临床诊治和流行病学监控提供有力工具。

Objective To establish a triple TaqMan qPCR method that can quickly distinguish and diagnose respiratory infections such as Chlamydia pneumoniae(Cpn),Chlamydia psittaci(Cps),and Chlamydia tracho-matis(Ct),which will contribute to the scientific prevention and control of Chlamydia and guide clinical medi-cation,providing guarantees for public health safety.Methods The specific genes ARGR,ompA and ORF8 in Cpn,Cps and Ct were selected to design the primers,establish the standard curve,optimize the reaction condi-tions and system,and verify the sensitivity and repeatability of the method.The accuracy of the real-time PCR method was verified by detecting three kinds of Chlamydia.Results The results showed that the established triple TaqMan qPCR method had good specificity and repeatability,and the sensitivity of detection was 45 cop-ies/μL(Cpn),40 copies/μL(Cps)and 43 copies/μL(Ct),respectively.A total of 600 clinical samples were tested with this method,and the detection rate was not significantly different from that of the reference meth-od(P>0.05),indicating that the method is reliable.Conclusion The triplex TaqMan qPCR assay established in this study has good specificity,repeatability,high sensitivity and reliable results for the detection of respira-tory Chlamydia,which provides a powerful tool for the clinical diagnosis,treatment and epidemiological sur-veillance of respiratory Chlamydia.