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A multiplex PCR (mPCR) protocol was developed for simultaneous detection of the gyrB gene in Streptococcus pneumoniae , Streptococcus mitis , and Streptococcus oralis , and the specificity was evaluated using 141 coccus strains. Genomic DNAs purified from S. pneumoniae , S. mitis , and S. oralis strains were efficiently detected with size differences, whereas no PCR products were amplified from any of the reference strains tested. A pilot study of 47 human oral swab specimens was conducted in parallel, and the mPCR assay identified S. pneumoniae in 1 sample, S. mitis in 8 samples, and S. oralis in 2 samples, providing a powerful means for characterization at the level of species compared with traditional culture analysis. Our results suggest that the mPCR protocol presented here is a sensitive and promising tool for the rapid detection and discrimination of S. pneumoniae , S. mitis , and S. oralis from clinical specimens.

We performed suppression subtractive hybridization to identify genomic differences between Streptococcus mitis and Streptococcus pneumoniae . Based on the pheA gene, a primer set specific to S. mitis detection was found in 18 out of 103 S. mitis -specific clones. Our findings would be useful for discrimination of S. mitis from other closely related cocci in the oral environment.