Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae.
A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 x 10(2) copies, corresponding to 2-20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100% was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.
Saito
Ryoichi
R
Department of Infection Control and Prevention, University of Tokyo Hospital, Bunkyo-ku, Tokyo 113-8655, Japan 2Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 3Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
Misawa
Yoshiki
Y
Department of Infection Control and Prevention, University of Tokyo Hospital, Bunkyo-ku, Tokyo 113-8655, Japan 2Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 3Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
Moriya
Kyoji
K
Department of Infection Control and Prevention, University of Tokyo Hospital, Bunkyo-ku, Tokyo 113-8655, Japan 2Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 3Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
Koike
Kazuhiko
K
Department of Infection Control and Prevention, University of Tokyo Hospital, Bunkyo-ku, Tokyo 113-8655, Japan 2Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 3Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
Ubukata
Kimiko
K
Department of Infection Control and Prevention, University of Tokyo Hospital, Bunkyo-ku, Tokyo 113-8655, Japan 2Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 3Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
Okamura
Noboru
N
Department of Infection Control and Prevention, University of Tokyo Hospital, Bunkyo-ku, Tokyo 113-8655, Japan 2Department of Microbiology and Immunology, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan 3Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan.
D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002675:Child, Preschool; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D009177:Mycoplasma pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D009305:Nasopharynx / Q000382:microbiology; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques; D011019:Pneumonia, Mycoplasma / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013696:Temperature
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes