Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Mycoplasma bovis

Abstract

A loop-mediated isothermal amplification (LAMP) assay targeting uvrC of Mycoplasma bovis was developed and evaluated. The assay specifically amplified only M. bovis; no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The sensitivity of the assay in pure cultures was 10-fold higher than that of polymerase chain reaction (PCR), with a detection limit of 34 CFU per reaction. The accuracy of the assay was further validated by both restriction analysis and nucleotide sequencing of the amplified product. The assay was applied to 98 specimens collected from cattle with respiratory disease or from healthy individuals and compared with a PCR-based assay. The sensitivity and specificity of the LAMP assay in terms of PCR was 100 and 74%, respectively. In conclusion, we successfully developed a rapid, specific, and sensitive LAMP test for M. bovis detection in a clinical setting.

Key words: Loop-mediated isothermal amplification, polymerase chain reaction, uvrC gene, Mycoplasma bovis, cattle.