多重PCR特异检测布鲁氏菌方法的建立

DEVELOPMENT OF A MULTIPLEX PCR FOR BRUCELLA DETECTION

目的建立一种快速检测布鲁氏菌的多重PCR方法,为临床快速检测、准确诊断布鲁氏菌提供依据。方法针对布鲁氏菌外膜蛋白31 kD、22 kD和2a基因,各设计一对引物,扩增片断大小分别为427 bp3、17 bp和830 bp。利用纯培养物和模拟标本,分析这3对引物的特异性和敏感性。结果3对引物均能特异扩增布鲁氏菌的DNA,31 kD、22 kD和2a,扩增纯培养物的敏感性分别为2.8×103、4.6×103和2.1×104cfu,将3对引物组合在一起建立了多重PCR方法,能从模拟的细胞和血液标本中扩增出特异的产物,对模拟的细胞感染标本的敏感性为1×105cfu,对模拟的血标本的敏感性为1.2×106cfu。结论针对外膜蛋白基因,建立了能特异地检测标本中布鲁氏菌的多重PCR检测方法。

Objective To develop a multiplex PCR for specific detection of Brucella. Methods According to the sequences of outer membrane protein gene（31 kD, 22 kD, and 2a）, three pairs of primers that amplify 427,317 and 830 bp of the corresponding gene respectively, were designed. Using Brucella culture and simulated samples, the specifit- ely and sensitivity of the PCR were analyzed. Results Three pairs of primer could specifically amplify Brucella DNA. The sensitivity of primer pairs of 31 kd, 22 kd, and 2a for bacteria culture was 2.8×10^3cfu, 4.6 ×10^3 cfu, and 2.1×10^4ctu respectively. By combining three primer pairs, a multiplex PCR was developed, the sensitivity of it for simulated cell culture of Brueella was 1 ×10^5cfu, and 1.2 ×10^6cfu for spiked blood sample. Conclusion Based on outer membrane protein genes, a multiplex PCR for specific detection of Brucella was developed.