Abstract
BackgroundMycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics.ResultsHere, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30 min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR.ConclusionsTo the author’s knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings.
Highlights
Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world
We developed a rapid, sensitive, and on-site recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) assay for the specific detection of M. bovis in the field
Evaluation of RPA nfo primer and probe sets The analytical specificity of seven primers combinations with LF-probes (Fig. 1 and Table 1) was confirmed using the genomic DNA extracted from M. bovis reference type strain PG45
Summary
Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. M. bovis has been confirmed as a major pathogen in bovine respiratory disease (BRD), but it has causes disease in cattle of all ages, such as arthritis, otitis media, mastitis, and reproductive disorders [1]. Due to lack of effectiveness of treatments for controlling the disease in affected herds and decreasing growth rate of the cattle, it can. In 2008, a severe cattle respiratory disease was reported in Hubei Province of China, thereafter, it quickly spread to over 11 Chinese provinces. The lack of an effective control methods to prevent the rapid spread of M. bovis as well as its stubborn persistence on farms requires rapid and accurate diagnosis when clinical signs first appear [1]
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