Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis

Abstract

Background: Mycoplasma 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.

Results: Here, 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.

Conclusions: To 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.

Keywords: Isothermal nucleic acid amplification; Lateral flow dipstick; Mycoplasma bovis; Rapid and visual detection; Recombinase polymerase amplification.

Fig. 1

Details (location and sequence) for each primer and LF probe set used in our RPA-LFD assay. a The target region spanned nucleotides 1464–1744 of the uvrC gene of Mycoplasma bovis (GenBank Accession: AF003959.1); b The target region spanned nucleotides 22–255 of the oppD-oppF gene of Mycoplasma bovis (GenBank Accession: AF130119.1)

Fig. 2

Screening of Mycoplasma bovis recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) primers and probe. a: The results of RPA-nfo reaction were detected by agarose-gel electrophoresis from seven set of primers and probe combination; b: ‘a’ showed results of RPA-nfo reaction by LFD test, and DNA template came from extracted Mycoplasma bovis reference type strain PG45 colonies, and ‘b’ was negative control (DNase-free water) that the corresponding combination of primers and probe. Samples were tested in triplicate with one reaction displayed in figure for each triplicate

Fig. 3

Optimization of incubation temperature and reaction time for Mycoplasma bovis recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay. a The amplification performance of RPA-LFD was effectively within the range of 35 °C to 45 °C. b Determination of amplification time. After 10 min of amplification reaction, the test line was clearly visible on the strip. Lane NC: negative control (DNase-free water). Samples were tested in triplicate with one reaction displayed in figure for each triplicate

Fig. 4

Comparison of sensitivities of the recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) and Real-time qPCR assays. Molecular sensitivity test results of the two assays were assessed using 10-fold serially diluted DNA as template. a Results by Real-time qPCR (with a detection limit of 83 copies/reaction DNA standards). b Results by RPA-LFD (with a detection limit of 20 copies/reaction DNA standards). Lane 1 to 8: 10-fold serially diluted Mycoplasma bovis plasmid DNA standards from 10⁷ to 10⁰ copies/uL. Lane NC: negative control (DNase-free water)