Establishment of a dual droplet digital PCR method for detecting the Brucella abortus A19-ΔVirB12 strains
Ruixue Xue, Zunfeng Chu, Linlin Xing, Zixin Jiang, Wenduo Jiang, Yingli Shang, Fangkun Wang, Hongmei Wang, Yuyu Zhang, Yuyu Zhang, Yanling Wang, Miao Yu, Xinglin Zhang, Mingjun Sun, Zouran Lan, Yue Zhang, Yue Zhang
Abstract
Brucellosis is a zoonosis that occurs worldwide, and vaccination is the main strategy for controlling it. In China, the Brucella abortus A19-ΔVirB12 strain is utilized in main vaccines. However, a high-sensitivity nucleic acid detection method to effectively differentiate Brucella infections from immunization with the A19-ΔVirB12 strain is lacking. Therefore, in this study, a duplex droplet digital PCR (ddPCR) assay was established using primers and probes targeting the VirB8 gene and the deleted VirB12 gene in the A19-ΔVirB12 strain. The specificity of the method was tested using genomic DNA of Mycobacterium bovis , Escherichia coli (O:157), Salmonella spp., Streptococcus spp., and A19-ΔVirB12 Brucella . Only A19-ΔVirB12 amplified VirB8 gene. The detection limits of the method for VirB8 and VirB12 were 2.13 × 10 0 and 2.26 × 10 0 copies/μL, respectively. In the detection of DNA in epidemic-related samples, the positive rate of ddPCR was much higher than that in the samples analyzed using the commercial fluorescence quantitative reagent kits. Meanwhile, the ddPCR of the A19-ΔVirB12 Brucella vaccine strain was identified in the clinical samples. In summary, the ddPCR method with high sensitivity and specificity was established, which will support the future identification of A19-ΔVirB12 Brucella vaccine strains in immunized and wild-type Brucella .