title: Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid.
abstract: Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.
authors.affiliation: Molecular Bacteriology Laboratory, International Vaccine Institute, Seoul, Republic of Korea.
authors.forename: Dong Wook
authors.initials: DW
authors.lastname: Kim
authors.forename: Paul Evan
authors.initials: PE
authors.lastname: Kilgore
authors.forename: Eun Jin
authors.initials: EJ
authors.lastname: Kim
authors.forename: Soon Ae
authors.initials: SA
authors.lastname: Kim
authors.forename: Dang Duc
authors.initials: DD
authors.lastname: Anh
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Seki
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D002555:Cerebrospinal Fluid / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D019741:Haemophilus influenzae type b / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008583:Meningitis, Haemophilus / Q000175:diagnosis* / Q000382:microbiology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D017931:DNA Primers