Open AccessDevelopment of a single‐tube loop‐mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis
Author(s) -
Huy Nguyen Tien,
Hang Le Thi Thuy,
Boamah Daniel,
Lan Nguyen Thi Phuong,
Thanh Phan,
Watanabe Kiwao,
Huong Vu Thi Thu,
Kikuchi Mihoko,
Ariyoshi Koya,
Morita Kouichi,
Hirayama Kenji
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/1574-6968.12002
Subject(s) - loop mediated isothermal amplification , biology , microbiology and biotechnology , streptococcus agalactiae , neisseria meningitidis , streptococcus suis , streptococcus pneumoniae , haemophilus influenzae , bacteria , escherichia coli , haeiii , pathogen , neisseria , streptococcus , polymerase chain reaction , gene , genetics , virulence , restriction fragment length polymorphism , dna , antibiotics
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 S treptococcus agalactiae and S treptococcus 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 16 S r RNA genes of the bacteria. The nucleotide sequences of the 16 S 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 S taphylococcus aureus , S treptococcus pneumoniae , S . suis and S . agalactiae . The broad range LAMP assay was highly specific without cross‐reactivity with other bacteria including H aemophilus influenzae , N eisseria meningitidis and E scherichia 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 D de I and H ae III .