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

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.