Establishment Of Recombinase Polymerase Amplification(RPA) For Detection Of Mycoplasma Bovis And The Method For Sample DNA Extraction

Mycoplasma bovis(M.bovis)is one of pathogens causing respiratory disease in cattle.And M.bovis can cause diseases such as mastitis,genital tract inflammation,otitis,arthritis,causing huge economic losses to the cattle industry worldwide.The aim of this study was to establish a method for rapid detection of M.bovis on site-Recombinase Polymerase Amplification(RPA)to provide technical support for the diagnosis of M.bovis.After screening the nucleotide sequences of M.bovis oppD/F and p80 gene,a method for detecting M.bovis LF-RPA was established based on the design of primers and probe for the p80 gene sequence(GenBank accession number:NZ_LQDV01000064.1).The optimal reaction condition is to put the mixture solution at 37?for 10 min.The specificity of the method was tested using the genome of the pathogen associated with bovine respiratory disease as the template,and the results showed no cross-reactivity with other pathogens.The sensitivity of the method was tested using the constructed p80 gene inserted plasmid and genome DNA as template,the results showed that the established LF-RPA can detect 38.9 copies/?L of plasmid and 5 pg/?L genome DNA.The coincidence rate between the LF-RPA and the conventional qPCR is 97.35%when applied to detect the DNA preserved in the laboratory.Extraction of genomic DNA is one of the key technical aspects of nucleic acid detection methods.Extraction efficiency and quality directly affect the sensitivity of detection methods.In order to screen out the extraction method suitable for direct extraction of M.bovis genomic DNA,the boiling method and an easy-going nucleic acid extraction method were compared with the commonly used DNA extraction kits in the laboratory.The extracted genomic DNA was evaluated for quality.The results show that the DNA was not pure as the one from the kit,but both methods are suitable for extracting genomic DNA from M.bovis culture for LF-RPA detection.The detection limit(LOD)of LF-RPA(49CFU)is lower than PCR(4.9×10~2 CFU)and qPCR(4.9×10~4 CFU)for boiling method.Similar results for the easy-going method is:the detection limits of LF-RPA(4.9×10~3 CFU)>PCR(4.9×10~4 CFU)>qPCR(4.9×10~5 CFU).The DNA amplification efficiency of boiling method is better than the easy-going extraction by LF-RPA for M.bovis culture.By adding the given amount of M.bovis into different normal clinic samples(milk,nasal swabs and lung tissues)from healthy cattel/cow,the simulated positive specimens were made for evaluation the DNA extraction method suitable for clinic samples.For milk and lung tissue samples,the DNA extracted with the easy-going method(the LOD for milk and lung samples is 50 CFU and 5×10~3 CFU,respectively)is more suitable for LF-RPA than that with boiling method(the LOD for milk and lung samples is 5×10~4 CFU and 5×10~7 CFU,respectively).For the nasal swabs,the better method for DNA extraction is boiling method(the LOD for LF-RPA is 5 CFU).Using the established LF-RPA and different DNA extraction methods,a total of 120 clinic samples was detected for M.bovis and the positive rate is 24.1%.The coincidence rate between the LF-RPA and the routine used qPCR(DNA extracted with the commercial bacterial gDNA extraction kit)in the lab is95%when applied to detect these samples.In conclusion,a point-of-care and on-site nucleotide detection method of LF-RPA for M.bovis was established in this study.The complete LF-RPA components include the different DNA extraction reagents/methods,RPA reaction reagents and LF test strip.This established LF-RPA will be a robust and helpful tool for detection and diagnosis of M.bovis infection.