奶牛隐性乳房炎乳房链球菌巢式PCR检测方法的建立

Establishment of Nested PCR of Streptococcus uberis for the Cow Subclinical Mastitis

为研究巢式PCR检测奶牛隐性乳房炎链球菌的灵敏度,以本实验室从新鲜奶样中分离并经过生理生化鉴定的乳房链球菌、无乳链球菌、大肠杆菌、金黄色葡萄球菌为研究对象,提取细菌DNA,采用巢式PCR技术,根据细菌16S rRNA序列保守区设计通用引物进行第1次PCR扩增,再在乳房链球菌16S rRNA保守区内的可变区设计特异引物对4种病原菌进行第2次PCR检测。结果表明:巢式PCR技术能检测出乳房链球菌16S rRNA保守区的可变区序列,与生理生化鉴定结果一致。该方法能快速有效检测出奶牛隐性乳房炎乳房链球菌。

In order to research sensitivity that detect cow subclinical mastiffs by nested PCR, Streptococcus uberis, Streptococcus agalactiae, Escherichia coli and Staphylococcus aureus separated from fresh dairy samples which have been identified by physiological and biochemical methods were used as tested materials. In this experiment, the bacterial DNA was extracted, then universal primer was designed according to bacteria 16S rRNA conserved region, nested PCR technique was used to amplify the nucleotide sequence. The specificity primers were designed based on various areas within conserved areas of the 16S rRNA genes of four pathogenic bacteria. The result showed nested PCR technique could detect various areas of conserved areas of the 16S rRNA genes of S. uberis. The result was the same as physiological and biochemical methods. This theory could be rapidly and efficiently applied in detecting S. uberis for the cow subclinical mastitis.