牛乳中副结核分枝杆菌套式PCR检测方法的建立

Development of nested PCR method for determination of Mycobacterium paratuberculosis in milk

目的建立牛乳中副结核分枝杆菌套式PCR检测方法,以便快速检测牛奶中副结核分枝杆菌。方法在制备的液体培养基中培养副结核分枝杆菌T、P18、P10,提取基因组DNA,应用DNAstar引物设计软件,根据副结核分枝杆菌独特的稳定性插入片段IS900设计内、外引物,建立检测牛乳中副结核分枝杆菌的套式PCR方法,并对该法的敏感性、特异性进行验证。结果用所建立的方法已成功扩增出目的基因,大小与预期一致;该法的敏感性为102个细菌/ml,特异性试验结果显示,除含副结核分枝杆菌的乳样为阳性外,含其他细菌的乳样均为阴性,表明本方法特异性强。结论已成功建立套式PCR检测方法,可用于牛乳中副结核分枝杆菌的检测。

Objective To develop a nested PCR method for rapid determination of Mycobacterium paratuberculosis in milk.Methods M.paratuberculosis was incubated in prepared liquid medium,from which genomic DNA was extracted.Internal and external primers were designed according to the sequence of specific inserted gene fragment IS900 of M.paratuberculosis by using DNAstar software,based on which a nested PCR method was developed and verified for sensitivity and specificity.Results Target gene was amplified successfully by the developed method,of which the length was consistent with that expected.The sensitivity of the developed method was 102 bacteria / ml.The test results for milk samples containing M.paratuberculosis by the method were positive,while those for samples containing other bacteria were negative,indicating high specificity of the method.Conclusion Nested PCR method was successfully developed,which might be used for determination of M.paratuberculosis in milk.