【作者】 齐洁；

【导师】 马红霞；

【作者基本信息】 吉林农业大学 ， 兽医硕士（专业学位）， 2021， 硕士

【摘要】 牛呼吸系统疾病(Bovine Respiratory Disease,BRD)主要病原耐药性的产生,给该病的预防和治疗造成了严重困扰,但其主要病原牛支原体(Mycoplasma bovis,M.bovis)、牛荚膜血清A型多杀性巴氏杆菌(Pasteurella mutocida,Pm)和牛溶血性曼氏杆菌(Mannheimia haemolytica,Mh)等均属苛养菌,实验室检测、药物敏感性检测等均耗时较长,无法有效的指导临床用药。为此,针对BRD主要病原,建立其主要病原鉴定及主要病原耐药基因、耐药靶位的快速、高通量检测的方法具有重要的临床意义。本研究基于荧光定量PCR,建立了BRD主要病原、常见耐药基因及药物靶位Taqman MGB探针检测芯片,为BRD快速诊断方法的建立奠定了基础。研究内容如下:首先,根据BRD主要病原基因组序列及耐药基因序列设计合成94条特异性探针及引物,镶嵌到固相载体中制成耐药基因及病原菌检测芯片,并对其特异性、敏感性、重复性进行评估。结果显示,构建的BRD主要病原及耐药基因检测芯片具有较好的特异性、重复性,相关系数均小于2%,标准差均小于0.5%;同时具有较好的敏感性,约1×10~1copies/μL。该检测方法可1h内完成临床样本检测,极大缩短了检测时间。其次,根据BRD主要病原氟喹诺酮类药物耐药决定区(Quinolone resistance determining region,QRDR)靶位突变特征,设计合成9条可检测QRDR常见突变的探针及引物序列,并对其特异性、敏感性、重复性进行测定。结果显示,构建的BRD主要病原QRDR药物及耐药基因检测芯片具有较好的敏感性,约1×10~4copies/μL,同时具有较好的特异性和重复性,相关系数均小于2%,标准差均小于0.5%。该检测方法可在1h内完成临床样本耐药靶位突变检测,极大缩短了检测时间。总之,本研究建立了可快速检测BRD主要病原、主要耐药基因及耐药靶位的检测方法,极大缩短了兽医临床BRD诊断及敏感药物筛选的时间,具有重要的实际意义。更多还原

【Abstract】 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.更多还原