实时荧光定量PCR快速诊断肺炎链球菌

Rapid detection of Streptococcus pneumoniae by real-time fluorescence quantitation PCR

目的建立实时荧光定量聚合酶链反应(PCR),用于肺炎链球菌检测和流行病学调查。方法以肺炎链球菌自溶素(lyt)和溶血素(ply)的基因为目的序列分别设计引物和探针,将其分别与10株肺炎链球菌、13株非肺炎链球菌DNA以及不同浓度梯度的肺炎链球菌DNA进行荧光定量PCR,并将引物和探针应用于200例临床标本检测,同时通过传统的微生物培养鉴定的方法来验证荧光定量PCR的特异性和敏感性。结果 10株肺炎链球菌均获得了明显的扩增产物,13株非肺炎链球菌DNA无明显的扩增信号,其检测敏感性可达100 fg;200例临床标本,实时荧光定量PCR检测出42例肺炎链球菌阳性(阳性率为21%),而培养法阳性16例(阳性率为8%)。结论实时荧光定量PCR是一种敏感、特异、快速的检测肺炎链球菌方法,可用于肺炎链球菌的诊断和流行病学调查。

Objective To establish real-time fluorescence quantitation polymerase chain reaction （PCR） for the detection and epidemiological studies of Streptococcus pneumoniae. Methods The autolysin （lyt） gene and hemolysin （ply） gene sequences were selected to develop primers and probe. A total of 10 strains of Streptococcus pneumoniae, 13 strains of non-Streptococcus pneumoniae DNA and the different concentration strains of Streptococcus pneumoniae DNA were detected by real-time fluorescence quantitation PCR, and 200 clinical specimens were detected by primers and probe. The specificity and sensitivity were also analyzed. Results The 10 strains of Streptococcus pneumoniae were measured to obtain amplification products. However, 13 strains of non-Streptococcus pneumoniae DNA had no obvious amplification, and the sensitivity was 100fg. Among the 200 clinical specimens, real-time fluorescence quantitation PCR detected 42 cases of Streptococcus pneumoniae-positive （the positive rate was 21% ）, while the culture method detected 16 cases of Streptococcus pneumoniae-positive （ the positive rate was 8% ）. Conclusions Real-time fluorescence quantitation PCR is a rapid, sensitive and specific assay for the detection of Streptococcus pneumoniae. It can be used for the diagnosis and epidemiological studies.