Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

Abstract

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 3 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.

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

“…Compared to a standard PCR protocol, LAMP requires a single bath at a constant temperature instead of a thermal cycler. The performances of LAMP-and PCR-based diagnostic systems, including real-time technologies (23,40), have been extensively compared. In general, LAMP was found to be either similar or superior to PCR, and more specific (e.g., 8,13,15), but a few studies proved otherwise, such as those reported by Kato et al (18), who showed that although LAMP was 10-fold more sensitive than standard PCR for the detection of the Clostridium difficile toxin B gene (tcdB), an optimized nested-PCR assay performed much better than LAMP.…”

“…(19,44) and pathogenic fungi such as Paracoccidioides brasiliensis (7) and Ochroconis gallopava (34) have also been addressed. LAMP-based approaches have been applied to a wide range of samples, such as paraffin-embedded tissues (7), whole blood (36), nasopharyn-geal swabs (17,40), dental plaques (23), eggs (13), and potato leaf samples (32).…”

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

“…The clinical samples for diagnosis are initially treated with a specifi c antibody-coated beads, and the target bacteria after reaction with their specifi c antibody are separated from other bacteria by immunomagnetic separation (IMS) [Bapanpally et al, 2014;Taguchi et al, 2007]. Loop-mediated isothermal amplifi cation (LAMP) is a sensitive, specifi c, convenient and fast nucleic acid amplifi cation technology, which obviates the need for expensive thermal cyclers used in conventional PCR [Saito et al, 2005]. The LAMP was designed with four specifi c primers for the six regions of the unique target gene using strand displacement DNA polymerase activity in addition to a nucleic acid amplifi cation activity at a constant temperature of 60-65°C [Hara- Kudo et al, 2008].…”

Polymerase Chain Reaction for Phytoplasmas Detection

“…using phytoplasma-specific antibodies and ELISA-based or lateral flow devices (LFD)-based systems, fall down because of a lack of sensitivity, and whilst a phytoplasma IgG antibody based system is commercially available for few phytoplasmas (103). Recently, Fera (Food and Environment Research Agency) developed isothermal amplification assays, such as the Loop-Mediated Isothermal Amplification (LAMP) procedure for detection of several human and plant pathogens including phytoplasmas (130,140,37,154). In the method the cycling accumulates stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops, and produces up to 10 9 copies of the target in less than 1 h at 65°C.…”