Abstract
This study aimed to detetct Mycoplasma bovis (M. bovis) in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the uvrC gene of M. bovis, an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for M. bovis without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 101 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the M. bovis genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect M. bovis in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to M. bovis infection causing bovine mastitis.
Highlights
As a major etiological agent of bovine mycoplasmosis globally, Mycoplasma bovis (M. bovis) causes various clinical symptoms in cattle, including pneumonia, arthritis, and mastitis (Nicholas and Ayling, 2003)
In the analytical specificity analysis, only the M. bovis DNA was amplified with the development of a typical fluorescence curve, and none of the other pathogens were amplified (Figure 1A), suggesting that the real-time recombinase polymerase amplification (RPA) assay was highly specific to M. bovis
The real-time RPA assay was conducted eight times, in which 1.0 × 107–1.0 × 101 copies of standard plasmid were detected in 8/8 runs, and 1.0 × 100, 0/8 (Figure 1B)
Summary
As a major etiological agent of bovine mycoplasmosis globally, Mycoplasma bovis (M. bovis) causes various clinical symptoms in cattle, including pneumonia, arthritis, and mastitis (Nicholas and Ayling, 2003). Serological methods are typically used as a herd-level disease diagnostic test, and a variety of commercial M. bovis ELISA tests have been developed to detect antibodies in the milk and serum (Heller et al, 1993; Le et al, 2002; Nicholas and Ayling, 2003). They are not ideally suited for M. bovis infection investigations with individual animals, as a misdiagnosis may occur due to the delayed seroconversion after natural infection (Calcutt et al, 2018). The high level of seroprevalence in many cattle herds restricts their routine use for diagnosis (Calcutt et al, 2018)
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