title: Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.
abstract: Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII.
authors.affiliation: Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, Japan.
authors.forename: Nguyen Tien
authors.initials: NT
authors.lastname: Huy
authors.forename: Le Thi Thuy
authors.initials: le TT
authors.lastname: Hang
authors.forename: Daniel
authors.initials: D
authors.lastname: Boamah
authors.forename: Nguyen Thi Phuong
authors.initials: NT
authors.lastname: Lan
authors.forename: Phan
authors.initials: P
authors.lastname: Van Thanh
authors.forename: Kiwao
authors.initials: K
authors.lastname: Watanabe
authors.forename: Vu Thi Thu
authors.initials: VT
authors.lastname: Huong
authors.forename: Mihoko
authors.initials: M
authors.lastname: Kikuchi
authors.forename: Koya
authors.initials: K
authors.lastname: Ariyoshi
authors.forename: Kouichi
authors.initials: K
authors.lastname: Morita
authors.forename: Kenji
authors.initials: K
authors.lastname: Hirayama
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D016920:Meningitis, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification; D013290:Streptococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013292:Streptococcus agalactiae / Q000235:genetics / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification; D017011:Streptococcus suis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S