多重荧光定量PCR方法鉴定布鲁氏菌属及牛羊种布鲁氏菌研究

Multiplex fluorescence quantitative PCR detection on Brucella spp., Brucella abortus,and Brucella melitensis

目的利用多重实时荧光PCR技术,建立快速鉴定布鲁氏菌的方法。方法根据布鲁氏菌特异性基因BCSP31,AlkB/IS711和BMEI1162/IS711的部分片段作为靶基因,分别设计探针引物,将扩增产物连接到PUCm18-T载体上,制备标准品及标准曲线,确定此方法的灵敏度。利用布鲁氏菌的其他生物型菌株和同源性较近的致病菌(汉赛巴尔通体、霍乱弧菌、土拉弗朗西斯菌、大肠杆菌O157∶H7、大肠杆菌O∶16、小肠结肠炎耶尔森菌O∶9、沙门氏菌N群血清型、嗜麦芽假单胞菌)验证所建立方法的特异性。通过布鲁氏菌标准菌株建立方法,优化体系后扩增97株地方株进行验证。结果应用多重Taq-Man荧光PCR技术检测布鲁氏菌结果显示,布鲁氏菌属、牛种布鲁氏菌和羊种布鲁氏菌有各自的荧光信号,而与其同源性较高的致病菌均未见荧光信号;由标准曲线可知该方法的单重和多重荧光PCR最低检测下限均约为102copy/μL(13～24fg/μL),是常规PCR灵敏度的100倍。结论建立的方法有灵敏度高、快速易操作等优点,可用于布鲁氏菌快速鉴定,并同时确定是否为牛种或羊种布鲁氏菌。

We applied real-time multiplex PCR to establish new and rapid identification methods for Brucella spp.Primers and probes were designed by target genes set from fragments of specific genes BCSP31,AlkB/IS711 and BMEI1162 / IS711 respectively.Amplification products were cloned into PUCm18-T vector for preparation of standard and standard curve to determine the detection limit.Specificity was verified by testing for other Brucella biotypes and pathogens with closer homology including Bartonella henselae,Vibrio cholerae,Francisella tularensis,Escherichia coli O157∶H7,Escherichia coli O∶16,Yersinia enterocolitica O∶9,group N Salmonella serotypes,and Pseudomonas maltophilia.The established method and optimized system for standard strain of Brucella spp.were then verified by amplification of 97 local strains.Results from multiplex TaqMan real-time PCR detection of Brucella showed that only Brucella spp.,Brucella abortus(B.abortus) and Brucella melitensis(B.melitensis) had respective fluorescence signal for each of them;while there was no fluorescent signal for pathogens with closer homology.According to standard curve,the lower limit of detection for both single and multiplex fluorescent PCR was 102copy/μL(13-24 fg/μL),100 times higher than that of conventional PCR.It's suggested that with advantages of higher sensitivity and fast and easy operation,the new method could be used to conduct rapid identification of Brucella spp.,B.abortus and B.melitensis in a single test.