目的 将从喜马拉雅旱獭全血中分离的布鲁氏菌利用聚合酶链式反应（PCR）技术进行种属鉴定。方法 将2016年收集的青海省喜马拉雅旱獭全血245份，分离血清，进行虎红平板凝集试验（RBT）和试管凝集试验（SAT）检测布鲁氏菌病抗体，将血清抗体阳性的喜马拉雅旱獭全血利用血培养法进行细菌培养，观察实验结果，分离、纯化疑似布鲁氏菌。将纯化的疑似布鲁氏菌进行涂片镜检及噬菌体裂解试验，同时提取核酸，制备模板，利用BCSP31-PCR、AMOS-PCR进行布鲁氏菌种型鉴定。结果 共分离出1株疑似布鲁氏菌，形态及染色特征与革兰阴性小杆菌相同。噬菌体裂解试验BK2裂解，Tb不裂解，可与单相特异性血清A、M凝集，确定为羊种生物Ⅲ型布鲁氏菌。BCSP31-PCR和AMOS-PCR试验琼脂糖凝胶电泳结果分别在223和731 bp处出现特异性条带，证实是羊种布鲁氏菌。结论 青海省首次从245份喜马拉雅旱獭全血中分离出1株布鲁氏菌并利用PCR试验证实是羊种布鲁氏菌。
Objective To perform species identification of Brucella isolated from the whole blood of Marmota himalayana by PCR. Methods A total of 245 whole blood samples were collected from M. himalayana in Qinghai province, China in 2016. Serum was isolated, and the Rose Bengal plate test and the tube agglutination test were used to detect brucellosis antibody. The blood culture method was used to perform bacterial culture of the whole blood samples of M. himalayana with positive antibody. The test results were observed and suspected Brucella strains were isolated and purified. Smear microscopy and the bacteriophage lysis test were performed for the purified suspected Brucella strains, nucleic acid was extracted to prepare templates, and then BCSP31-PCR and AMOS-PCR were used to identify Brucella strains. Results One suspected Brucella strain was isolated with the same morphology and staining characteristics as Gram-negative bacilli. The bacteriophage lysis test showed lysis of BK2 and non-lysis of Tb, and the strain could agglutinate with the single-phase specific serum A and M; therefore, the strain was identified as Brucella melitensis type Ⅲ. BCSP31-PCR agarose gel electrophoresis showed a specific target band at 223 bp, and AMOS-PCR agarose gel electrophoresis showed a specific target band at 731 bp; the strain was proved to be B. melitensis. Conclusion A Brucella strain is isolated for the first time from 245 whole blood samples of M. himalayana and is confirmed to be B. melitensis by PCR in Qinghai province.
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