利用扩增片段长度多态性分析鉴别炭疽和蜡样芽孢杆菌

Identification of the Bacillus anthracis and B.cereus Using Amplified Fragment Length Polymorphism Analysis

目的：利用扩增片段长度多态性（AFLP）分析建立鉴别炭疽芽孢杆菌和蜡样芽孢杆菌的分子生物学方法。方法：3株炭疽芽孢杆菌和3株蜡样芽孢杆菌基因组经限制性内切酶EcoRⅠ和MseⅠ酶切后与对应接头连接,通过预扩增和选择性扩增获得特异性DNA片段,将片段进行毛细管电泳,并利用GeneScan和BioNumerics软件对电泳数据进行分析。结果：选择性扩增最佳引物组合为EcoRⅠ-G/MseⅠ-A,其扩增片段在100~500 bp范围内的有效数量为40~50条;比较炭疽芽孢杆菌和蜡样芽孢杆菌的AFLP特征峰值图和DNA指纹图谱,确定了5个有明显差异的片段区。结论：利用AFLP分析可对芽孢杆菌属中相近的炭疽芽孢杆菌和蜡样芽孢杆菌进行鉴别,该方法可作为炭疽芽孢杆菌传统鉴定方法的补充。

Objective： To identify the Bacillus anthracis and B.cereus by amplified fragment length polymorphism （AFLP） analysis. Methods： 3 different isolates of B.anthracis and 3 different isolates of B.cereus were used in this study. The genomic DNA was digested with EcoR I and Mse 2, and the resulting fragments were ligated to double-stranded adapters. The digested and ligated DNA was then pre-amplified and selective-amplified by PCR. The samples were analyzed on an ABI PRISM 3100 and the AFLP profiles were analyzed using GeneScan and BioNumerics analysis software. Results： The best-quality primer combinations was EcoR I-G/Mse I-A, the AFLP fingerprint for an isolate was represented by about 40-50 fragments between 100 and 500 bp, and there had 5 visible specific markers between B.anthracis and B.cereus strains in AFLP fingerprint. Conclusion： The B.anthracis and B.cereus isolates were identified by AFLP analysis and this molecular biology method could application with the classical biology method.