牛肉和牛奶中布氏杆菌免疫磁珠富集方法的建立

Enrichment of Brucella from Beef and Milk by Immunomagnetic Beads

为建立牛肉和牛奶样品中布氏杆菌的免疫磁珠富集方法,采用布氏杆菌特异性抗体包被磁珠并优化包被条件,制备成免疫磁珠。对不同稀释梯度的布氏杆菌菌液用免疫磁珠富集,联合布氏杆菌选择性平板培养或荧光PCR,确定免疫磁珠的敏感性。同时,分别用免疫磁珠富集添加有其它细菌的布氏杆菌,富集后于选择性平板培养,统计目标菌落数并挑取可疑目标菌落用荧光PCR鉴定,确定免疫磁珠的特异性。此外,将该方法应用于人工模拟牛肉和牛奶样品中布氏杆菌的富集,进一步确定免疫磁珠的富集效果。结果表明:在1mL布氏杆菌菌液中,2 mg免疫磁珠富集联合选择性平板培养能检测≥2×10~3CFU布氏杆菌,其敏感性高于免疫磁珠联合荧光PCR法。在其它细菌的高浓度背景下,免疫磁珠仍能够特异性地富集到布氏杆菌。此外,用该免疫磁珠富集方法可成功地富集到人工模拟牛肉和牛奶样品中的布氏杆菌。表明建立的免疫磁珠富集方法,可用于实际检测时牛肉和牛奶样品中布氏杆菌的富集,具有良好的应用前景。

To establish a method of enriching Brucella by immunomagnetic beads from beef and milk samples,coating the magnetic beads with the specific antibody of Brucella,several factors of coupling were optimized and the immunomagnetic beads were prepared.To determine the sensitivity of the immunomagnetic beads,it was used to enrich Brucella in a series of dilution solutions,which were associated with culture in Brucella selectively medium or real-time PCR.To determine the specificity of the immunomagnetic beads,it was used to enrich Brucella from the Brucella mixture with other non-Brucella bacteria respectively.Then the bacteria enriched by immunommagnetic beads were cultured in Brucella selectively medium.The target colonies were counted and the suspicious target colonies were identified by real-time PCR.Moreover,the method for enrichment of Brucella based on immunomagnetic beads was applied to capture Brucella from artificially spiked beef and milk samples to confirm the enrichment effect of immunomagnetic beads.The results showed that 2 mg immunomagnetic beads with Brucella selective medium could enrich the target bacteria in Brucella solution of which the minimum dilution was 2×10~3/mL.The sensitivity of the immunomagnetic beads with Brucella selective medium was higher than the immunomagnetic beads with real-time PCR.Brucella could also be enriched specifically by the immunomagnetic beads under the conditions of high concentration other non-Brucella bacteria.In addition,the application of capture Brucella from artificially spiked beef and milk samples by the immunomagnetic beads was successful.These results indicated that the established method for enrichment of Brucella based on immunomagnetic beads can be used for enriching Brucella in beef and milk samples in practical detection.This method has good prospects.