布鲁菌双重TaqMan荧光定量PCR检测方法的建立及初步应用

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中图分类号：R378.5；S852.61 文献标志码：A 文章编号：0366-6964（2026）01-0369-09

Abstract：This study aimed to establish a cost-effective duplex TaqMan real-time quantitative PCR （qPCR） assay for the rapid detection of Brucella infection in animals and animal products，and to differentiate B.abortus from other Brucella species. This method provides a scientifically valid diagnostic tool for distinguishing currently used vaccine strains and novel vaccines under development. Specific primers and TaqMan probes targeting the galU and Omp3l genes were designed based on GenBank sequences. Recombinant plasmids were constructed. The assay's specificity，sensitivity， and repeatability were rigorously evaluated. Clinical serum and tissue samples from animals with brucellosis were tested. A duplex TaqMan qPCR assay for Brucella detection was successfully developed. The limit of detection（LOD） for recombinant plasmid standards was 1.93×10ocopies⋅μL-1 The assay exhibited no cross-reactivity with other Gram-negative bacteria.Both intra-assay and interassay reproducibility were excellent，with coefficients of variation （CV） consistently below 2% ： Testing of 156 samples （including laboratory-prepared and clinical specimens） revealed a positive agreement rate of 69.75% compared to BCSP31-PCR. Furthermore，the duplex TaqMan qPCR demonstrated significantly higher sensitivity （ 100% ）：than conventional PCR （69.75% ）andthe RBPT 58.65% ）. The established duplex TaqMan qPCR assay provides a robust technical foundation for the clinical differential diagnosis，epidemiological surveillance，and eradication programs of brucellosis.

Keywords： Brucella;duplex TaqMan real-time PCR；detection method;differential diagnosis （  Corresponding authors： YANG Yanling，E-mail： m1804321339@163. com; CHEN Si， E-mail ： 374582512@qq.com

布鲁菌病（简称布病）是由布鲁菌感染引起的一种流行广泛、危害较大的人兽共患病之一。（剩余14256字）