肺炎链球菌多重巢式荧光PCR分型方法的建立和应用

Establishment and application of a multiplex real-time PCR for serotyping of Streptococcus pneumoniae

目的建立一种敏感且节省标本的多重巢式荧光PCR方法,用于临床标本中肺炎链球菌的分型。方法以检测肺炎链球菌种属(lytA基因)和20组常见血清型的引物和探针为基础,分别采用92株不同血清型的肺炎链球菌菌株和系列稀释的参考DNA模板,建立多重巢式荧光PCR方法。评价该方法的特异性和敏感性,同时与荧光PCR方法进行比较。将该方法用于14份肺炎链球菌培养阳性和30份肺炎链球菌培养阴性的临床标本的检测,评价实际应用效果。结果多重巢式荧光PCR与荧光PCR方法能准确鉴定/区分大部分菌株(90/92)的血清型。前者的检测灵敏度为1~100 fg/μl,9种血清型的灵敏度高于荧光PCR方法(10~100 fg/μl)。44份临床标本中,多重巢式荧光PCR与荧光PCR方法分别检测出34和31份阳性(P=0.778),2种方法的DNA模板使用量分别为15μl和66μl。结论多重巢式荧光PCR比荧光PCR方法节省标本且更加灵敏。

Objective To establish a sensitive and sample-saving multiplex real-time PCR assay(MRT-PCR) for serotyping of Streptococcus pneumoniae isolated from clinical samples. Methods The MRT-PCR assay included 21 sets of primers and probes(one for S. pneumoniae species and 20 for serotypes). Comparative analysis of MRT-PCR and the direct real-time PCR was performed to evaluate the specificity, sensitivity, and efficacy of each assay by serotyping 92 well-characterized clinical isolates of S. pneumoniae and 44 clinical samples(including 14 positive samples and 30 negative samples). Results The MRT-PCR assay can identify/distinguish the serotypes of most strains of S. pneumoniae as real-time PCR did(90/92). The lower limit of detection was 1-100 fg/μl of bacterial genomic DNA for MRT-PCR, for most serotypes, it was lower than that for real-time PCR. MRT-PCR detect the serotypes of 77%(34/44) of samples with 15 μl of extracted DNA, while real-time PCR detect the serotypes of 70%(31/44) with 66 μl of extracted DNA(P=0.778). Conclusion The MRT-PCR assay is a sample-saving assay for the specific and sensitive serotyping of clinical S. pneumoniae isolates.