多重荧光定量聚合酶链反应对呼吸道感染患儿支原体和衣原体感染的诊断价值

目的 探讨多重荧光定量PCR技术(TaqMan探针法)在检测呼吸道感染住院患儿中肺炎支原体(MP)、肺炎衣原体(CP)和沙眼衣原体(CT)的应用价值.方法 构建质粒标准品,梯度稀释后进行扩增建立标准曲线检测多重荧光定量PCR方法的扩增效率,并验证其敏感性、特异性和重复性;收集151例疑似呼吸道感染住院患儿的咽拭子样本,采用多重荧光定量PCR技术检测MP、CP和CT基因片段,其中128例样本同时进行MP-IgM和CP-IgM的血清学检测,评价该方法在临床检测中的应用价值.结果 本实验中MP、CP和CT三种病原体的引物和探针的扩增效率分别达到109.20％、116.90％和112.80％,三种病原体的最低检出浓度分别为50、25和25 pg/μL,与其他已知病原体不存在交叉反应;不同批次间三种病原体的扩增Ct值的变异系数均小于3％.151份样本中,多重PCR检测出MP、CP和CT阳性率分别为23.84％(36份)、17.88％(27份)和2.65％(4份),128份样本中MP-IgM阳性检出率为22.65％;CP-IgM阳性检出率为14.06％.结论 多重荧光定量PCR方法可以为临床幼儿呼吸道感染病原体的早期检测提供快速可靠的辅助诊断依据.

Diagnostic value of multiplex real-time polymerase chain reaction for mycoplasma and trachomatis infection in respiratory tract infection children

Objective To investigate the diagnostic value of multiplex real-time PCR (TaqMan probe) for simultaneous detection of Mycoplasma pneumoniae (MP),Chlamydophila pneumonia (CP) and Chlamydia trachomatis (CT) in hospitalized children with respiratory tract infection.Methods The standard curves were plotted based on recombinant plasmids that was amplified after gradient dilution,so as to measure the amplification efficiency of the multiplx real-time PCR method,and the sensitivity,specificity and reproducibility were verified.There were 151 throat swab samples from hospitalized children with suspected respiratory tract infection collected.The MP,CP and CT gene fragments were detected by multiplex real-time PCR.There were 128 samples identified by MP-IgM and CP-IgM serological detection to evaluate the diagnostic value in clinical detection.Results The amplification efficiency of MP,CP and CT primers and probes reached up to 109.20％,116.90％ and 112.80％;and the minimum detectable concentration were 50,25 and 25 pg/μL,respectively.No cross reactions with other known pathogens was observed,and coefficients of variation among different amplification reactions were all less than 3％.In 151 samples,the positive rates of MP,CP and CT by multiplex real-time PCR were 23.84％ (36 samples),17.88％(27 samples) and 2.65％(4 samples).In 128 samples,the positive rates of MP-IgM and CP-IgM were 22.65％ and 14.06％,respectively.Conclusions The multiplex real-time PCR method can provide rapid and reliable evidence for early pathogen detection in children with respiratory tract infection.