探针荧光定量PCR在肺炎链球菌检测中的应用

Clinical application of real-time fluorescence quantitive PCR for detecting Streptococcus pneumoniae

目的建立探针荧光定量PCR检测肺炎链球菌的方法。方法针对肺炎链球菌种属特异性基因lytA,设计合成了特异引物和探针,研究引物和探针的灵敏度和特异性,确定循环阈值(cycle threshold,ct)的临界值(cut-off value)。将荧光定量PCR和细菌培养法进行比较,同时检测158份肺炎患者痰液标本加以验证。结果针对LytA基因所设计的引物和探针能灵敏地检出常见致病的血清型肺炎链球菌株,检测灵敏度为每个反应100个基因组DNA拷贝。通过荧光量PCR方法,35株肺炎链球菌中检测结果为阳性有34株,检测结果为阴性的有1株;15株非肺炎链球菌全部为阴性。通过荧光定量PCR方法,158份痰液标本中共检测出34份肺炎链球菌阳性,其中10份培养出相应的病原菌。肺炎链球菌阳性患者的白细胞数量和住院时间均显著高于阴性患者(P<0.05)。肺炎链球菌阳性患者住院时间均显著长于阴性患者(P<0.05)。结论探针荧光定量PCR方法能特异地检测肺炎链球菌,具有很高的灵敏度,能提高临床肺炎链球菌感染患者标本的阳性检出率。

Objective To establish an assay for the detection of Streptococcus pneumoniae by real-time fluorescence quantititive polymerase chain reaction (PCR).Methods Special primers and probe for the autolysin A (lytA)gene were designed.The sensitivity and specificity of primers and probe were studied,and cut-off of cycle threshold was assayed.158 clinical specimens were confirmed by real-time fluorescence quantitative PCR and bacterial culture method.Results Primer and probe design for LytA gene could sensitively detect serotype Streptococcus pneumoniae strains of common pathogenic,and the sensitivity was 100 copies.Among 35 strains of Streptococcus pneumoniae,34 cases were detected to be positive for Streptococcus pneumoniae by real-time fluorescence quantitative PCR,while 1 case was detected to be negative;among 15 strains of non-Streptococcus pneumoniae, all were detected to be negative.Among the 158 clinical sputum specimens,34 cases with Streptococcus pneumoniae were detected by real-time fluorescence quantitative PCR,while only 10 cases with Streptococcus pneumoniae were detected by the culture method.White blood cells count and time in hospital of cases with Streptococcus pneumoniae were higher than those of cases without Streptococcus pneumoniae (P <0.05 ). Conclusion Real-time fluorescence quantitative PCR is a sensitive and specific assay for the detection of Streptococcus pneumoniae.It can be used for the diagnosis of Streptococcus pneumoniae.