生鲜乳中布鲁氏杆菌多重PCR检测方法的建立

Establishment of multiplex PCR assay for Brucella in fresh and raw milk

为了建立快速、准确检测生鲜乳中布鲁氏杆菌的方法,试验采用细菌基因组提取试剂盒提取生鲜乳中的布鲁氏杆菌基因组DNA,针对布鲁氏杆菌保守抗原Omp31、Omp25设计引物,并设计细菌16S r DNA基因序列作为内参引物,通过引物浓度和退火温度的调整优化,建立了生鲜乳中布鲁氏杆菌的多重PCR检测方法。结果表明：建立的多重PCR检测结果与预期结果一致,可以扩增出布鲁氏杆菌Omp31（472 bp）、Omp25（140 bp）特异性片段以及细菌16S r DNA的通用片段。而对于其他致病菌只能扩增出1 500 bp的细菌16S r DNA通用片段,具有较强的特异性。该多重PCR检测方法灵敏度可达1×10^5cfu/m L,较单一PCR检测方法灵敏度有所下降。说明本试验建立的检测方法可完成生鲜乳中布鲁氏杆菌的快速检测。

The aim of the present study was to establish a rapid and accurate method for detecting Brucella in fresh and raw milk. In this study,genomic DNA of Brucella in fresh and raw milk was extracted by bacterial genomic extraction kit. Primers were designed for Brucella Omp31,Omp25,and the bacterial 16 S r DNA gene sequence was designed as an internal reference primer. Based on the optimization of primer concentration and annealing temperature,a multiplex PCR method for detecting Brucella in fresh and raw milk was established. The results showed that the multiplex PCR could be used to amplify the specific fragments of Brucella Omp31（472 bp）,Omp25（140 bp） and 16 S r DNA. For other bacteria,only 1 500 bp fragment of bacterial r DNA gene could be amplified by using this PCR method. It was indicated that the method had strong specificity.The sensitivity of this multiplex PCR was lower than that of single PCR,and the sensitivity was up to 1×105 cfu/m L. The detection method established in this study could be completed within 4 h for rapid detection of Brucella in fresh and raw milk.