In order to detect Bacterium burgeri in clinical milk samples,the nested PCR for Omp31 gene was established. The ex-traction method of the genomic DNA of Bacterium burgeri was improved and the nested PCR reaction sets were optimized by orthogo-nal test. The first optimized PCR reaction condition:annealing temperature 56.4 ℃,Mg2+concentration 1.5 mmol·L-1,the rTag DNA polymerase 0.2 μL,Primer 0.3 uM,dNTP concentration 0.2 mM. The second PCR optimization condition:annealing temperature 53.3℃,Mg2+concentration 1.5 mmol·L-1,the DNA polymerase 0.25 μL,Primer 0.4 uM and dNTP concentration 0.1 mM. The sensi-tivity of nested PCR was 8 CFU·mL-1 which was 1 000 times higher than bacterial isolate method. The specificity test showed that there was no cross reaction with Escherichia coli,Staphylococcus aureus,Yeast,Bacillus and Klebsiella. Compared with test tube ag-glutination,the positive coincidence rate was 100% and the negative coincidence rate was 86.36%. This nested PCR assay could be applied to detect the Bacterium burgeri in milk.%为检测牛奶中的布鲁氏菌，通过对布鲁氏菌外膜蛋白31 kDa的一段基因进行套式PCR扩增，建立从临床奶样中检测布鲁氏菌的方法。改进了奶样中布鲁氏菌基因组DNA的提取方法，通过正交试验的方法优化了套式PCR试验的反应条件，优化后的一扩PCR反应条件为：退火温度56.4℃，Mg2+浓度1.5 mM，rTaq酶0.2μL，引物0.3μM，dNTP浓度0.2 mM；二扩PCR反应条件为：退火温度53.3℃，Mg2+浓度1.5 mM，rTaq酶0.25μL，引物0.4μM，dNTP浓度0.1 mM。其敏感性为8 CFU·mL-1，比细菌分离试验敏感1000倍；与大肠杆菌、金黄色葡萄球菌、酵母菌、隐秘杆菌和克雷伯氏菌均无交叉反应；与试管凝集试验检测布鲁氏菌病的阳性符合率是100%，阴性符合率是86.36%。试验建立的套式PCR方法可用于检测奶样中布鲁氏菌。
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