Study on the detection method of Brucella differential type PCR

Currently, the prevention and control of zoonotic diseases such as brucellosis in some developing countries still primarily rely on "vaccination + quarantine and culling." However, the inability to differentiate between natural infection and vaccine-induced antibodies in immunized livestock has become a new challenge in veterinary clinical diagnosis. To address the issue of distinguishing between wild-type Brucella strains and attenuated vaccine strains in vaccinated animals, this study developed singleplex and multiplex PCR assays targeting six differential loci (NC7250, GL2189, wbkF-wbkD, WP788.1, eryC, and BP26). The primer concentrations and reaction conditions were optimized, ultimately determining the optimal primer concentration ratio as 2:1:1:4:2:4 and the optimal annealing temperature as 60 °C. The results demonstrated that the singleplex PCR exhibited excellent specificity, accurately distinguishing between different Brucella species as well as the A19 and S19 vaccine strains. The hexaplex PCR yielded negative results for non-target bacteria such as Streptococcus and Salmonella. The detection sensitivities were as follows: 1.99 × 10⁻¹ ng/μL for S2 strain DNA, 2 × 10⁻¹ ng/μL for Rev.1 strain DNA, and 2 × 10⁰ ng/μL for S19 strain DNA. The singleplex and hexaplex PCR methods targeting six genes established in this study enable the simultaneous differentiation of wild-type Brucella strains from commonly used vaccine strains, effectively addressing the clinical gap in diagnostic techniques for strain discrimination. This provides a scientific basis for veterinary quarantine, disease eradication, and vaccine selection strategies.