Development and validation of a loop-mediated isothermal amplification assay for the detection of Mycoplasma bovis in mastitic milk
Mycoplasma mastitis is often difficult to control due to a lack of rapid and accurate diagnostic tools. The aim of the current study was to develop a loop-mediated isothermal amplification (LAMP) assay for the detection of Mycoplasma bovis (M. bovis) in mastitic milk. The assay was developed using primers designed for three different target genes: uvrC, 16S rRNA, and gyrB, and validated using mastitic milk samples previously found positive for the target pathogen. Specificity of the developed assay was determined by testing cross-reactivity of LAMP primers against closely related bovine mastitis bacterial pathogens. The sensitivity was found to be higher compared to conventional polymerase chain reaction (PCR). The LAMP assay was also capable of detecting M. bovis in PCR-negative milk samples of cows with clinical mastitis. The uvrC primers were found to be more sensitive, while gyrB primers were more specific; however, 16S rRNA primers were less specific and sensitive compared to either uvrC or gyrB primers. Cohen’s kappa values for uvrC, gyrB, and 16S rRNA primers used in the LAMP assays were 0.940, 0.970, and 0.807, respectively. There was a high level of agreement between the test results and the true-disease status as indicated by the receiver operating characteristic (ROC) curve. Our findings suggest that the newly developed LAMP assays targeting the uvrC and gyrB genes could be a useful tool for rapid and accurate diagnosis of mastitis caused by M. bovis.
We are thankful to Dr. Peter Thomson, School of Life and Environmental Sciences, The University of Sydney, Australia, for providing assistance in statistical analysis. Special thanks to Quality Milk Production Services (QMP), Cornell University, Ithaca, NY, USA, for providing standard bacterial strains and mastitic milk samples.
AA carried out the study and wrote the manuscript; MI, MT, WS, TY, and CNM conceived and designed the experiments; MI and YFC supervised the work; MI and WS did proofreading of the paper; all the authors read and approved the final manuscript.
This work was supported by the Higher Education Commission of Pakistan under the International Research Support Initiative Program (IRSIP) grant #266.22.
Compliance with ethical standards
The authors declare that they have no competing interests.
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