Real-time PCR assays for the detection and quantification of Streptococcus pneumoniae

Streptococcus pneumoniae is the main etiologic agent of pneumonia worldwide. Because the members of the viridans group streptococci share a high degree of DNA sequence homologies, phenotypic and genotypic discriminations of S. pneumoniae from the viridans group are difficult. A quantitative real-time PCR assay targeting the capsular polysaccharide biosynthesis gene (cpsA) was developed as a species-specific detection tool for S. pneumoniae. The specificity was evaluated using genomic DNAs extracted from 135 oral cocci strains. Twenty-seven S. pneumoniae strains tested positive, whereas 108 other strains including Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis did not show a specific signal. The linear regression of standard curves indicated high correlations between the log numbers of S. pneumoniae cells and the C(T) values (R(2)=0.99). The minimal limit of detection was 32 fg of purified genomic DNA, equivalent to 14 genomes of S. pneumoniae. This new real-time PCR method may be very useful as a rapid and specific tool for detecting and quantifying S. pneumoniae.