A Novel DNA Amplification Method for Haemophilus influenzae Detection

OBJECTIVES: Haemophilus influenzae is a common major human pathogen associated with community-acquired pneumonia, bactermia, meningitis, and otitis media. While traditional antimicrobial therapy has proven to be an effective treatment, the emergence of antimicrobial-resistant strains has resulted in an increasing number of cases. Sensitive and specific assays that can be promptly completed in the clinical laboratory are essential for early diagnosis and effective antibiotic therapy. However, isolation and identification of this strain is complicated by contamination with normal flora. It has been difficult to separate H. influenzae from the ecologically and genotypically similar species H. parainfluenzae which is a commensal of the human oral cavity. Recently, a novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was developed. Its product can be recognized by checking turbidity in the reaction tube with the naked eye. The purpose of this study is to examine the applicability of the method to detect H. influenzae. METHODS: We designed species-specific LAMP primer sets to identify H. influenzae by targeting the gene encoding the outer membrane protein. The specificity and the sensitivity of LAMP primers were compared to that by the conventional PCR. The amplification speed of LAMP was examined using a loopamp realtime turbidimeter, which was a real-time monitor regarding the turbidity of the solution. RESULTS: (i) the LAMP primers designed in this study were specific to H. influenzae; (ii) the sensitivity of this method was over 10 times higher than that of the conventional PCR method; (iii) the detection of H. influenzae from clinical samples with this method was also examined and we confirmed its clinical usefulness. CONCLUSION: These results suggested that this method may be a speedy, easy to use, sensitive, and reliable means of diagnosing H. influenzae infection.