驼乳中布鲁氏菌实时荧光PCR快速检测方法的建立

Establishment of a real-time fluorescent PCR assay for rapid detection of Brucella in camel milk

中文关键词:  驼乳  布鲁氏菌  实时荧光PCR

英文关键词:camel milk  Brucella  real-time fluorescence PCR

中文摘要:

      目的 建立驼乳中布鲁氏菌的实时荧光PCR快速检测方法。方法 根据布鲁氏菌外膜蛋白omp10基因的碱基序列, 设计引物探针, 从模拟布鲁氏菌污染的驼乳中提取布鲁氏菌DNA片段进行实时荧光PCR扩增检测。结果 该方法从布鲁氏菌M5株中扩增出了特异性目的片段omp10, 而对大肠埃希式杆菌等细菌的扩增结果均为阴性。经过10倍倍比稀释的模板进行检测, 该方法的灵敏度为最低可检出2.31×10?4 ng/μL的DNA。标准曲线循环阈值与模板浓度呈良好线性关系, 相关系数为0.9928, 扩增效率E=0.97。结论 建立的实时荧光PCR检测方法具有特异性强、灵敏度高的优点, 为驼乳中布鲁氏菌病的早期预防及流行病学监测提供了有效的技术手段。

英文摘要:

      Objective To establish a rapid detection method for Brucella in camel milk by real-time fluorescent PCR method. Methods According to the base sequence of the Brucella outer membrane protein omp10 gene, a primer probe was designed to extract the Brucella DNA fragment from camel milk simulating Brucella contamination for real-time fluorescent PCR amplification detection. Results The specific target fragment omp10 was amplified from Brucella M5, but the amplification results for bacteria such as Escherichia coli were negative. This method had a minimum sensitivity of 2.31×10?4 ng/μL DNA when tested with a template diluted 10-fold. The circulation threshold of the standard curve showed a good linear relationship with the template concentration, with the correlation coefficient of 0.9928 and amplification efficiency E=0.97. Conclusion The established real-time PCR detection method has the advantages of strong specificity and high sensitivity, and provides an effective technical means for early prevention and epidemiological monitoring of brucellosis in camel milk.