肺炎链球菌TaqMan实时荧光定量PCR的建立及临床应用

Establishment and Application of TaqMan Real-Time PCR assay for Diagnosis of Streptococcus Pneumoniae

目的：建立TaqMan实时荧光定量PCR检测肺炎链球菌的方法，应用于临床儿童社区获得性肺炎（CAP）标本的快速筛查。方法根据 GenBank公布的肺炎链球菌自溶酶基因（lytA）设计引物和探针，建立实时荧光定量 PCR，并对体系进行优化；同时以痰培养法做双盲对照，应用于1504份 CAP患儿标本检测。结果 TaqMan实时荧光定量 PCR菌液的最低检出限可达18.75 cfu/PCR，无交叉反应，特异度好；对1504份儿童CAP标本检测，141份肺炎链球菌实时荧光定量PCR阳性，其中140份肺炎链球菌痰培养阳性，该方法灵敏度为100％，特异度为99.93％。从样品处理到结果报告仅需2.5 h。结论 TaqMan实时荧光定量PCR快速、简便、灵敏度高、特异度强，可用于儿童CAP中肺炎链球菌的初筛和指导抗生素的的合理使用。

Objective To develop TaqMan real-time PCR assay for detection and identification of streptococcus pneumonia iso-lated from children CAP.Methods Based on the sequences of lytA gene,primers and probe were designed and the assay was optimized.Then 1 504 sputum samples were detected by culture and the developed assay with double-blind testing.Results The lower limit of detection in the developed real-time PCR assay was 18.75 cfu/PCR,and had no cross reaction.141 strep-tococcus pneumonia strains were detected from 1 504 samples and 140 were isolated by culture.The whole process just nee-ded 2.5 h.Conclusion The established assay is rapid,simple,high sensitivity and specificity.It is not only valuable for the i-dentification of streptococcus pneumonia,but also provide evidence for antibiotic therapy.