Development Of Drug Resistance Gene And Drug Target Detection Chip For Main Pathogens Of Bovine Respiratory Diseases

Bovine Respiratory Disease(Bovine Respiratory diseases,BRD)main pathogen resistance,for the prevention and treatment of the Disease caused serious problems,but its main pathogenic Mycoplasma cattle(Mycoplasma bovis,M.bovis),Bovine serum type A capsule kill sex Pasteurella(Pasteurella mutocida,Pm)and hemolytic mann’s coli(Mannheimia haemolytica,Mh)all belong to fastidous bacteria,such as Laboratory testing and drug sensitivity testing are time-consuming,which cannot effectively guide clinical drug use.Against BRD main pathogens,therefore,the main pathogen identification and drug resistance genes,resistance to targeted the establishment of the rapid,high-throughput detection method has important clinical significance,therefore,this study based on the fluorescence quantitative PCR,established the BRD main pathogens,common drug resistance gene and drug targeted Taqman MGB probe detection chip,laid a solid foundation for establishment of BRD rapid diagnostic method.The research contents are as follows:First of all,94 specific probe and primer sequences were designed and synthesized according to the main pathogen genome sequences and drug-resistant gene sequences of BRD,and then inserted into solid phase vector to make drug-resistant gene and pathogen detection chips,and their specificity,sensitivity and repeatability were evaluated.The results showed that the main BRD pathogens and drug resistance gene detection chips constructed had good specificity and reproducibility,with standard deviations less than 0.5%and correlation coefficients less than 2%.Good sensitivity1×10~1copies/μl;Clinical sample detection can be completed within 1h,greatly reducing the detection time.Based on the characteristics of QRDR target mutation,nine probes and primers were designed and synthesized,and the specificity,sensitivity and repeatability of these primers were analyzed.The results showed that the constructed microchips for the detection of QRDR drugs and drug resistance gene of BRD had good sensitivity of1×10~4copies/μL.It had good specificity and reproducibility,with standard deviations less than 0.5%and correlation coefficients less than 2%.Drug resistance target mutation detection of clinical samples can be completed within 1h,greatly shortening the detection time.In conclusion,this study established a detection method that can quickly detect the main pathogens,major drug-resistant genes and drug-resistant targets of BRD,laying a foundation for the rapid diagnosis of veterinary clinical BRD and the rapid determination of drug resistance.