Detection of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus agalactiae using 16S rRNA gene / 대한임상병리학회지

ABSTRACT

BACKGROUND: Delayed treatment of acute bacterial meningitis often causes death or serious neurological defects. Rapid and accurate diagnosis is very important for effective treatment. Although the Gram stain and latex agglutination test for major causative bacteria, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus agalactiae, are useful for early detection of acute bacterial meningitis, their sensitivity is not satisfactory. In this study, we purpose to develop a PCR strategy for the simultaneous detection of N. meningitidis, H. influenzae, S. pneumoniae and S. agalactiae. METHODS: Primers were designed from the 16S rRNA genes of N. meningitidis, H. influenzae, S. pneumoniae and S. agalactiae, which were constituted with 3 senses and 5 antisenses, and 7 primer pairs. The PCR assay was divided into two steps, the first PCR resulted in a general bacterial amplicon with universal primers, and the second were performed with species specific primer pairs in four combination reaction tubes. PCR sensitivity and specificity were tested to clinical isolates of 4 N. meningitidis, 3 H. influenzae, 14 S. pneumoniae, 6 S. agalactiae, 14 Staphylococcus epidermidis, and 25 reference strains of different species. RESULTS: The species specific primer pairs showed specific DNA amplification to all clinical isolates tested. All 25 reference strains of different species and S. epidermidis were distinguished from N. meningitidis, H. influenzae, S. pneumoniae and S. agalactiae except for Pseudomonas aeruginosa, Neisseria and Candida species, The PCR detection limit for S. agalactiae was 200 CFU. CONCLUSIONS: The seminested PCR using bacterial 16S rRNA gene was sensitive and specific for the detection of Neisseria species, H. influenzae, S. pneumoniae and S. agalactiae. This results warranted the application of this method to cerebrospinal fluids to diagnose bacterial meningitis.