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Published on: June 25, 2020
Detection of Haemophilus influenzae by loop-mediated isothermal amplification (LAMP) of the outer membrane protein P6
Hirotaka Torigoe1, Mitsuko Seki, Yoshihisa Yamashita
1Department of Oral Health Sciences, Nihon University School of Dentistry, Tokyo 101-8310, Japan.
Abstract:
It is difficult and time-consuming to distinguish Haemophilus influenzae from the genotypically similar Haemophilus parainfluenzae, which is a commensal of the human oral cavity. The novel nucleic acid amplification technique of loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was evaluated for H. influenzae detection. A H. influenzae-specific LAMP primer set was designed for the outer membrane protein P6 gene. Primer set specificity was validated using 4 Haemophilus spp. and 13 other species. Within 60 min, LAMP detected 100 or more copies of purified DNA with a sensitivity that was 10-fold higher than that of conventional PCR. This method can be used to differentiate H. influenzae from H. parainfluenzae strains. Thus, LAMP may represent a sensitive and reliable means of diagnosing H. influenzae infection.
Insights
Loop-mediated isothermal amplification (LAMP) offers a rapid and sensitive method for detecting Haemophilus influenzae. This technique effectively distinguishes it from Haemophilus parainfluenzae, aiding in infection diagnosis.
Area of Science:
- Microbiology
- Molecular Biology
- Diagnostic Technologies
Background:
- Distinguishing Haemophilus influenzae from the similar Haemophilus parainfluenzae is challenging and time-consuming.
- Haemophilus parainfluenzae is a common commensal organism found in the human oral cavity.
- Accurate identification of H. influenzae is crucial for diagnosing infections.
Purpose of the Study:
- To evaluate the efficacy of loop-mediated isothermal amplification (LAMP) for the specific detection of Haemophilus influenzae.
- To assess LAMP's ability to differentiate H. influenzae from H. parainfluenzae.
- To determine if LAMP offers a more rapid and sensitive diagnostic method compared to conventional techniques.
Main Methods:
- Designed a Haemophilus influenzae-specific LAMP primer set targeting the outer membrane protein P6 gene.
- Validated primer set specificity against four Haemophilus species and thirteen other bacterial species.
- Assessed LAMP sensitivity and compared it with conventional PCR using purified DNA.
Main Results:
- LAMP demonstrated high specificity in detecting H. influenzae.
- The method achieved a sensitivity of 100 or more DNA copies within 60 minutes.
- LAMP exhibited a 10-fold higher sensitivity than conventional PCR.
- The technique successfully differentiated H. influenzae from H. parainfluenzae strains.
Conclusions:
- Loop-mediated isothermal amplification (LAMP) is a sensitive and rapid nucleic acid amplification technique.
- LAMP provides a reliable method for the specific detection and differentiation of Haemophilus influenzae.
- This approach holds promise for improving the diagnosis of H. influenzae infections.
