Development of a TaqMan MGB-probe based real-time fluorescence quantitative PCR assay for rapid detection of Brucella

The new generation TaqMan Minor Groove Binding(MGB) probe approach was used to develop the specific and sensitive real time fluorescence quantitative PCR(RTFQ-PCR) assay for rapid detecting Brucella in our study.The specific primers and probe for TaqMan MGB-probe based RTFQ-PCR were designed based on 16S rRNA sequence of genus Brucella.A TaqMan MGB-probe based RTFQ-PCR assay was established,and its specificity,sensitivity and stability were assessed.Then,the established TaqMan MGB-probe based RTFQ-PCR assay was applied to detect Brucella in 773 animal specimens during 2008-2010,and compared with conventional PCR assay.The specificity of this established TaqMan MGB-probe based RTFQ-PCR was high and there were no cross-reactivity with Yersinia enterocolitica,Yersinia pseudotuberculosis,Salmonella enterica,Escherichia coli,Pseudomonas aeruginosa,Campylobacter jejuni,and Clostridium piliforme.The correlation coefficient and slope value of standard curve were 0.999 and-3.301 respectively and the efficiency of TaqMan MGB-probe based RTFQ-PCR was 100.872%.The TaqMan MGB-probe based RTFQ-PCR assay was able to accurately detect Brucella DNA from brucellosis-positive specimens.The detection limit for this assay was 9.3 copies,and the sensitivity of this assay was 100-fold higher than conventional PCR assay.The TaqMan MGB-probe based RTFQ-PCR was preformed to detect Brucella in 773 animal specimens,and a total of 53 specimens were positive for Brucella.However,there was only 37 specimens were positive by conventional PCR.The results showed that TaqMan MGB-probe based RTFQ-PCR for Brucella was more sensitive than conventional PCR assay,and it could detect Brucella DNA from animal specimens directly,and detection time is only 2 hours.To the knowledge of the authors,this is the first TaqMan MGB-probe based RTFQ-PCR assay for the direct detection of Brucella in animal specimens.The technique appears to be sufficiently adaptable to meet the needs of rapid detecting requirements to identify infectious pathogens.