荧光定量PCR检测肺炎链球菌感染方法的建立

目的 建立Taqman探针法荧光定量PCR检测肺炎链球菌感染的实验室诊断方法.方法 针对肺炎链球菌自溶素基因设计引物及探针,建立荧光定量PCR Taqman探针法检测肺炎链球菌感染.采用肺炎链球菌标准株,构建质粒,用质粒构建标准品,扩增标准品,绘制标准曲线并检验新建荧光定量PCR的敏感性,采用本实验冻存其他菌DNA样本,检测其特异性;采集2015年9月至2015年12月份北京地区家5家医院儿童门诊或病房,诊断为呼吸道感染患儿的咽拭子标本133例,对新建荧光定量PCR法进行临床样本的验证.结果 绘制了荧光定量PCR Taqman探针法检测肺炎链菌感染的标准曲线;新建荧光定量PCR可检测的肺炎链球菌最低拷贝数为10copies/μL;新建荧光定量PCR可成功区分几种病原体DNA,具有较好特异性;采用新建荧光定量PCR检测临床样本132例,其中肺炎链球菌阳性25例,阳性率为18.94%.结论 新建立的荧光定量PCR检测肺炎链球菌感染的实验室诊断方法具有较高的敏感性和特异性,可用于临床样本的检验.

Developing a fluorescent quantitative PCR method for detection of streptococcus pneumonia infection

Objective To develop a Taqman probe fluorescence quantitative PCR assay for detection of streptococcus pneumonia (SP). Methods Primer and probe were designed according to SP autolysin and fluorescence quantitative PCR Taqman probe were established to detect SP infection.The standard plasmids extracted from SP standard strains were used to constitute and amplify standard samples.The sensitivity of the fluorescence quantitative PCR assay was tested by established standard curves.The specificity of fluorescence quantitative PCR assay was tested by the DNA samples of other bacteria frozen in the laboratory.Meanwhile, 133 throat swab samples from inpatients and outpatients children diagnosed as respiratory infection were collected from 5 hospitals in Beijing area from September 2015 to December 2015 to validate the fluorescence quantitative PCR assay.Results The standard curve of fluorescence quantitative PCR Taqman assay detecting SP was made.This newly-developed method could detect at least 10 copies of SP, and could successfully distinguish several DNAs of the pathogens with good specificity.Newly developed fluorescence quantitative PCR was used to detect 132 clinical samples, including 25 positive SP with positive rate of 18.94%.Conclusion The fluorescence quantitative PCR assay is of great sensitivity and specificity, and it can be widely used for the detection of SP.