奶牛乳房炎6种主要致病菌多重PCR检测方法的建立及初步应用

Establishment and application of multiplex PCR method for detection of six main pathogens of dairy cow mastitis

为了同时检测奶牛乳房炎中的6种主要致病菌,本试验分别针对金黄色葡萄球菌的nuc基因、无乳链球菌的ef-tu基因、绿脓杆菌的eta基因、停乳链球菌的16srRNA基因、致病性大肠杆菌的Phoa基因、牛支原体的opp D/F基因设计合成了6对特异性引物。在建立并验证单一PCR的基础上,构建多重PCR反应体系,对其反应体系进行优化,验证了该方法的特异性和敏感性。结果表明,该6重PCR检测方法对奶牛乳房炎的其他病原菌没有特异性扩增,对牛支原体、致病性大肠杆菌、停乳链球菌、绿脓杆菌、无乳链球菌、金黄色葡萄球菌最低检出终浓度分别为1.1×10^(5)、1.41×10^(6)、1.6×10^(3)、3×10^(5)、3.8×10^(4)和7.3×10^(3)拷贝/μL。对采集的宁夏地区98份临床乳房炎病例乳样的检测结果显示:牛支原体、致病性大肠杆菌、停乳链球菌、绿脓杆菌、无乳链球菌、金黄色葡萄球菌的检出率分别为10.2%(10/98)、14.2%(14/98)、14.2%(14/98)、12.2%(12/98)、18.3%(18/98)、18.3%(18/98)。本研究建立的多重PCR检测方法具有良好的特异性及敏感性,可用于奶牛乳房炎6种主要致病菌的检测及流行病学调查。

In order to simultaneously detect six major pathogenic bacteria in dairy cow mastitis,six pairs of specific primers were designed for nuc gene of Staphylococcus aureus,ef-tu gene of Streptococcus agalactiae,eta gene of Pseudomonas aeruginosa,16srRNA gene of Streptococcus agalactiae,Phoa gene of pathogenic Escherichia coli and oppD/F gene of Mycoplasma bovis.Based on the establishment and verification of single PCR,the multiplex PCR reaction system was constructed and optimized,which verified the specificity and sensitivity of this method.The results showed that the multiplex PCR detection method had no specific amplification to other pathogenic bacteria of cow mastitis,and the lowest detectable concentrations of Mycoplasma bovis,Pathogenic Escherichia coli,Streptococcus alactiae,Pseudomonas aeruginosa,Streptococcus agalactiae,Staphylococcus aureus were1.1×10^(5)copies/μL,1.41×10^(3)copies/μL,1.6×10^(3)copies/μL,3×10^(5)copies/μL,3.8×10^(4)copies/μL and 7.3×10^(3)copies/μL respectively.The detection results of milk samples collected from 98 clinical mastitis cases in Ningxia showed that the detection results of Mycoplasma bovis,Pathogenic Escherichia coli,Streptococcus alactiae,Pseudomonas aeruginosa,Streptococcus agalactiae,Staphylococcus aureus were 10.2%(10/98),14.2%(14/98),14.2%(14/98)and 12.2%(12/98),18.3%(18/98),18.3%(18/98).The results showed that the multiplex PCR detection method established in this study had good specificity and sensitivity,and the detection method could be used for the detection and epidemiological investigation of six main pathogenic bacteria of dairy cow mastitis.