Inverted dot blot hybridization 검색법을 이용한 mitis 군 연쇄상구균 종-특이 DNA 프로브 및 PCR 프라이머 개발

Abstract

The purpose of this study was to develope of species-specific DNA probes of mitis group streptococci (MGS) and species-specific PCR primers base on the nucleotide sequences of the DNA probes. A collection of 95 strains consisting of 81 clinical isolates, 8 reference strains and 6 type strains of MGS were examined. The identification of 81 clinical isolates was performed by phylogenetic analysis using multilocus sequence typing (MLST) method using three housekeeping genes, 16S rRNA gene (16S rDNA), RNA polymerase beta-subunit encoding gene (rpoB) and manganese superoxide dismutase gene (sodA). The HindⅢ-digested genomic DNAs from the type strains were cloned by random cloning method. To screen the species-specific probes of Streptococcus mitis, S. pneumoniae, S. oralis, S. parasanguinis, S. sanguinis and S. gordonii, IDBH were performed and confirmed the specificity of the DNA probes by southern blot analysis method. The species-specific PCR primers were designed base on the nucleotide sequences of the species-specific DNA probes. The MLST data revealed that 93% of clinical isolates were classified into 11 species of MGS by rpoB gene. SodA gene is not adequate for the identification of S. mitis and S. pneumoniae and did not coincide with the results obtained by the rpoB gene. The data showed that the Sgo41, Sor24, Spa17 and Ssa21 DNA probes were species-specific for S. gordonii, S. oralis, S. parasanguinis, and S. sanguinis, respectively. Interestingly, the strain-specific probes for Streptococcus mitis KCTC 3556T, Smi21 DNA probe, was developed. The PCR data showed that species-specific PCR primer for S. gordonii, Sgo41-F1/R1. and two pairs of S. mitis KCTC 3556T-specific primers, Smi21-f-F2/R2 and Smi21-b-F/R were developed. These results indicate that Sgo41-F1/R1 primer could be useful in the identification of S. gordonii and this primer is suitable for the detection of S. gordonii in clinical samples. In addition, two PCR primer sets, Smi21-f-F2/R2 and Smi21-b-F/R, have proven useful for the identification of S. mitis KCTC 3556T, especially with regard to the maintenance of the strain.