肺炎链球菌多重巢式荧光PCR分型方法的建立和应用

袁梦; 李马超; 胡鹏威; 徐丽; 高源; 段永翔; 朱兵清; 邵祝军

doi:10.3784/j.issn.1003-9961.2020.04.014

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

摘要

摘要:
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15 μl和66 μl。
结论多重巢式荧光PCR比荧光PCR方法节省标本且更加灵敏。

Abstract:
ObjectiveTo establish a sensitive and sample-saving multiplex real-time PCR assay (MRT-PCR) for serotyping of Streptococcus pneumoniae isolated from clinical samples.
MethodsThe 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).
ResultsThe 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).
ConclusionThe MRT-PCR assay is a sample-saving assay for the specific and sensitive serotyping of clinical S. pneumoniae isolates.

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