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 × 102 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.
ChiaW. K.,
SpenceL.,
DunkleyL.,
BradburyW.1988; Development of urease conjugated enzyme-linked immunosorbent assays (ELISA) for the detection of IgM and IgG antibodies against Mycoplasma pneumoniae in human sera. Diagn Microbiol Infect Dis 11:101–107[CrossRef]
Dorigo-ZetsmaJ. W.,
ZaatS. A.,
Wertheim-van DillenP. M.,
SpanjaardL.,
RijntjesJ.,
Van WaverenG.,
JensenJ. S.,
AnguloA. F.,
DankertJ.1999; Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children. J Clin Microbiol 37:14–17
FedorkoD. P.,
EmeryD. D.,
FranklinS. M.,
CongdonD. D.1995; Evaluation of a rapid enzyme immunoassay system for serologic diagnosis of Mycoplasma pneumoniae infection. Diagn Microbiol Infect Dis 23:85–88[CrossRef]
FoyH. M.1993; Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. Clin Infect Dis 17 (Suppl. 1):S37–S47
HarrisR.,
MarmionB. P.,
VarkanisG.,
KokT.,
LunnB.,
MartinJ.1988; Laboratory diagnosis of Mycoplasma pneumoniae infection.2. Comparison of methods for the direct detection of specific antigen or nucleic acid sequences in respiratory exudates. Epidemiol Infect 101:685–694[CrossRef]
HongT. C.,
MaiQ. L.,
CuongD. V.,
ParidaM.,
MinekawaH.,
NotomiT.,
HasebeF.,
MoritaK.2004; Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol 42:1956–1961[CrossRef]
IevenM.,
UrsiD.,
Van BeverH.,
QuintW.,
NiestersH. G.,
GoossensH.1996; Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M.pneumoniae in acute respiratory tract infections in pediatric patients. J Infect Dis 173:1445–1452[CrossRef]
IwamotoT.,
SonobeT.,
HayashiK.2003; Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M.avium , and M. intracellulare in sputum samples. J Clin Microbiol 41:2616–2622[CrossRef]
KokT. W.,
VarkanisG.,
MarmionB. P.,
MartinJ.,
EstermanA.1988; Laboratory diagnosis of Mycoplasma pneumoniae infection.1. Direct detection of antigen in respiratory exudates by enzyme immunoassay. Epidemiol Infect 101:669–684[CrossRef]
TempletonK. E.,
ScheltingaS. A.,
GraffelmanA. W. & 7 other authors; 2003; Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae
. J Clin Microbiol 41:4366–4371[CrossRef]
TjhieJ. H.,
Van KuppeveldF. J.,
RoosendaalR.,
MelchersW. J.,
GordijnR.,
MacLarenD. M.,
WalboomersJ. M.,
MeijerC. J.,
van den BruleA. J.1994; Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J Clin Microbiol 32:11–16
UrsiD.,
DirvenK.,
LoensK.,
IevenM.,
GoossensH.2003; Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control. J Microbiol Methods 55:149–153[CrossRef]