Analysis Of The Microbial Diversity In Bovine Nasal Cavity And Establishment Of RPA Detection Method For Mycoplasma Bovis

Mycoplasma bovis(M.bovis)is one of the main pathogen causing pneumonia in cattle.In addition,M bovis can also cause a variety of diseases such as mastitis,arthritis and meningitis,which contributes to the dire threat of livestock production.In the process of how to control the spread of the above diseases and reduce the economic losses caused by them,the rapid and accurate detection ofM.bovis is crucial,16S rRN A amplicon sequencing and metagenomic sequencing were used to determine the composition and abundance of nasal microorganisms in cattle suffering from respiratory diseases,to determine the infection status of M.bovis,and to use recombinant polymerase amplification technology(RPA)to establish detection method of M.bovis,it has positive significance for the early discovery,early detection,early isolation,early treatment and prevention and control of diseases such as cattle respiratory disease,and it can provide technical support for the healthy development of animal husbandry.In our study,through the analysis of 16S rRNA amplicome sequencing and metagenomic sequencing of clinical samples from ningxia region,it was confirmed that M.bovis is a key pathogen in cattle respiratory diseases;three RPA detection methods were established including M.bovis Basic RPA,nfo RPA and exo RPA,we detected the clinical samples using the three methods.The results are shown as following:(1)Blindly picking cattle nose swabs(18 samples)with respiratory diseases in 5 large-scale farms in Ningxia,and taking healthy cattle nose swabs(11 samples)as the control group,16S rRNA diversity analysis found that the disease group and the healthy group have significant differences in the composition of bovine nasal microbes.The main pathogenic microorganisms which make the diseased group different from the healthy group are Mycoplasma.(2)Metagenomic sequencing was performed on the above samples,and through KRONA analysis,it was clear that the pathogenic microorganism that caused the main difference between the disease group and the healthy group was M.bovis.(3)Bioinformatics analysis combined with PCR technology,8 specific marker genes,including LppA,were preliminarily screened for detection of M.bovis.(4)The M.bovis Basic RPA detection method based on the LppA gene was established.The reaction was performed at 39℃ for 20 minutes,and detection was performed by agarose gel electrophoresis.This method has good specificity,has no cross-reaction with other related pathogenic bacteria that cause bovine pneumonia,the minimum detection limit of the sensitivity test is 6× 101 copies/μL,and it has appropriate intra-and inter-assay repeatability.(5)The M.bovis nfo RPA detection method based on the LppA gene was established.The reaction can be detected by lateral flow chromatography test strip at a temperature of 39℃ for 10 minutes.This method has good specificity,the minimum detection limit of the sensitivity test is 3×101 copies/μL,and it has appropriate intra-and inter-assay repeatability.(6)The M.bovis exo RPA detection method based on LppA gene.After 15 minutes reaction at 39℃,it can be detected by T16-ISO constant temperature fluorescence amplification detector,with good specificity and lowest sensitivity test.The limit is 6×102 copies/μL,which has appropriate repeatability within and between batches.(7)A total of 118 clinical samples of nose swabs from ningxia region were collected,and the three detection methods of M.bovis established in this study were used to test the clinical samples,and the test results were compared with the PCR detection results.It was found that the positive rate of PCR detection was 28%,and the positive rates of Basic RPA was 29%,nfo RPA was 35%and exo RPA was 33%,respectively,and the coincidence rates with the PCR were 97%,92%and 94%.In summary,this study identified significant differences in species composition between healthy bovine nasal microorganisms and bovine nasal microorganisms with respiratory diseases,and M.bovis was the main species that caused this difference;Three detection methods including Basic RPA,nfo RPA and exo RPA were established to detect M.bovis.These detection methods have good specificity,sensitivity and repeatability and the characteristics are that they are rapid,easy and do not need to use large scale equipment,which can provide technical support for the detection of M.bovis and the prevention and control of mycoplasma bovine disease.