Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis

• Published in: FEMS microbiology letters 2012-12, Vol.337 (1), p.25-30
• Main Authors: Huy, Nguyen Tien, Hang, Le Thi Thuy, Boamah, Daniel, Lan, Nguyen Thi Phuong, Van Thanh, Phan, Watanabe, Kiwao, Huong, Vu Thi Thu, Kikuchi, Mihoko, Ariyoshi, Koya, Morita, Kouichi, Hirayama, Kenji
• Format: Article
• Language: English
• Subjects: assay; Assaying; Bacteria; Bacteriological Techniques - methods; Bacteriology; Biological and medical sciences; Cross-reactivity; diagnosis; DNA Primers - genetics; DNA, Bacterial - genetics; DNA, Ribosomal - genetics; E coli; Endonuclease; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene sequencing; Genes; Haemophilus; Humans; LAMP; Meningitis; Meningitis, Bacterial - diagnosis; Meningitis, Bacterial - microbiology; Microbiology; Miscellaneous; Molecular Diagnostic Techniques - methods; Nucleic Acid Amplification Techniques - methods; Nucleotides; Pathogens; Primers; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sensitivity and Specificity; simultaneous; Species; Staphylococcal Infections - diagnosis; Staphylococcal Infections - microbiology; Staphylococcus; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; Streptococcal Infections - diagnosis; Streptococcal Infections - microbiology; Streptococcus agalactiae; Streptococcus agalactiae - genetics; Streptococcus agalactiae - isolation & purification; Streptococcus infections; Streptococcus pneumoniae - genetics; Streptococcus pneumoniae - isolation & purification; Streptococcus suis; Streptococcus suis - genetics; Streptococcus suis - isolation & purification
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/1574-6968.12002

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.