奶样无乳链球菌PCR检测方法敏感性研究

Study on sensitivity of PCR assay for presence of S. agalactiae of milk samples

意义无乳链球菌是引起牛临床或亚临床乳腺炎主要病原菌,研究PCR奶样检测方法的敏感性,对探索病原无乳链球菌的简易、快速诊断具有重要意义.目的通过对含细菌数不同奶样的PCR扩增试验的观察和比较研究,探索其检测方法的敏感度.方法常规检测方法初选奶样,然后对其病原菌奶样进行培养、增菌和生化鉴定,筛选出牛无乳链球菌.将得到的无乳链球菌按10个稀释度奶样进行稀释,以标准该菌株作为阳性对照,分别进行PCR扩增、电泳.结果基于编码16S rRNA亚基的编码基因序列设计引物的PCR方法具有独特敏感性,可以检测到1ml生奶样中的一个细菌.

Bovine mastitis caused by Streptococcus agalactiae is mainly sub-clinical infection.It is significant to explore the methods to identify S.agalactiae easily and early,and to study the sensitivity of the Polymerise chain reaction(PCR) assay on the basis of DNA sequences coding for bacterium 16S rRNA with the milk samples.A sensitivity test is implemented by using a series of milk samples to which are added different concentrations,10-fold dilution of S.agalactiae colony-forming units,and is determined by the dilution factor in the test tube of highest dilution for which a recognizable amount of PCR product is obtained.When amplification follows directly on addition of the S.agalactiae dilution to the milk samples,products are obtained at a concentration of 104 to 105 cfu/ml or greater.When amplification follows a selective enrichment period,products are obtained at an initial concentration of 1 cfu/ml.