奶牛乳腺炎致病菌三重qPCR检测方法的建立OA

To establish a rapid detection method for the main pathogenic bacteria of bovine mastitis,in this study,specific primers and probes were designed and synthesized based on the conserved regions of the phoA gene of Escherichia coli,the mdh gene of Klebsiella pneumoniae,the femA gene of Staphylococcus aureus,and the internal reference gene ACtB,and a triple fluorescence PCR detection method for Esche-richia coli,Staphylococcus aureus and Klebsiella pneumoniae in bovine mastitis was established.This method can specifically detect three common pathogenic bacteria of bovine mastitis,and has no cross-reac-tion with common pathogens such as Streptococcus agalactiae,Streptococcus dysgalactiae,Mycoplasma bovis and Salmonella,showing strong specificity.The minimum detection limits for phoA,mdh and femA were 16.9,16.7 and 20.4 copies/μL respectively,and the intra-group and inter-group coefficient of variation were both less than 2％,indicating good repeatability.This method was used to detect 90 raw milk samples of bovine mastitis,and the detection results were consistent with those of the single fluorescence PCR method.The results show that the established triple fluorescence quantitative PCR detection method for Escherichia coli,Klebsiella pneumoniae and Staphylococcus aureus in bovine mastitis has the characteris-tics of strong specificity,high sensitivity and simple operation,and is suitable for clinical diagnosis and de-tection of bovine mastitis.