鸭疫里默氏杆菌巢式PCR检测方法的建立

Establishment of Nested PCR for Detection of Riemerella anatipestifer

为建立一种更加准确、敏感的鸭疫里默氏杆菌检测方法,根据鸭疫里默氏杆菌16S rRNA基因序列设计2对巢式PCR引物,在完成最佳反应条件筛选、特异性、敏感性、重复性试验的基础上,建立巢式PCR检测方法。结果表明,建立的巢式PCR对1、2、10、X型鸭疫里默氏杆菌的16SrRNA都能扩增出特异性片段,而对大肠杆菌、多杀性巴氏杆菌、沙门菌、鸭源金黄色葡萄球菌的基因组不能扩增出特异性片段,DNA最小检测量为0.392fg/μL,是常规PCR的1000倍。该巢式PCR方法具有快速、敏感、特异、通用的优点,可用于鸭疫里默氏杆菌感染的检测、分子流行病学调查和分离菌株的快速鉴定。

To establish a more exact and sensitive method to detect Riemerella anatipestifer, two pairs of nested PCR primer were designed according to Riemerella Anatipestifer 16 S rRNA sequence. The optimum reaction conditions were screened, and sensitivity, specificity and repeatability of nested PCR had carried out. The results showed that Riemerella aAnatipestifer of types 1,2, 10, X could be amplified by nested PCR, and Escherichia coli, Pasteurella multocida, Salmonella, Staphylococcus aureus could not be amplified. The minimum amount of detected DNA was 0.392 fg/μL and nested PCR was 1 000 times more sensitive than conventional PCR. The nested PCR assay was rapid, sensitive, specific and universal, which provided a greatly improved tool for diagnosis, molecular epidemiology survey and identification of Riemerella anatipestifer.