Loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) for detection of Mycoplasma bovis

Abstract:

[Background]Mycoplasma bovis is one of the major pathogens causing bovine mastitis, arthritis, and respiratory diseases, seriously endangering the healthy development of the cattle industry. [Objective] To establish a convenient, rapid, sensitive, and specific method based on loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) for the detection of M.bovis. [Methods] The conserved region was selected by comparison of the P48 specific sequence of M.bovis and used to design primers by Primer Explorer V5 online. The designed primers were compared with the LAMP primers designed with oppD/F,oppD, and urvC as target genes by the fluorescent dye method. The optimal internal primers (FIP/BIP) designed based on P48 were labeled with biotin and 6-carboxyfluorescein, respectively. The reaction temperature, time, and primer concentration ratio were optimized by the single factor method. LAMP was combined with LFD for the detection of M.bovis. Finally, the sensitivity, specificity, repeatability, and clinical application effect of the established method were evaluated. [Results] The LAMP primers designed based on P48 had stronger fluorescence signal, lower Ct value, and higher amplification efficiency, outperforming the reported LAMP primers. The reaction conditions were optimized as 60 ℃, primer concentration ratio (F3/B3:FIP/BIP) of 1:4, and 40 min. The lower limit of detection of the established method was 17.28 fg/μL, which was 1 000 times lower than that of the PCR method, the standard method in the industry. There was no cross-reaction with 9 pathogens causing bovine respiratory diseases, such as Pasteurella multocida and bovine herpes virus (BHV). The results of inter-batch and intra-batch tests were consistent. The detection rate of 39 clinical nasal swabs by the established method was 28.21%, which was higher than that (23.07%) of the PCR method. [Conclusion] A sensitive, specific, and easy-to-use LAMP-LFD method for detecting M.bovis was successfully established, which provided technical support for the prevention and control of M.bovis.