Establishment of a dual real-time fluorescent PCR method for detecting the Brucella abortus A19-△VirB12 mutant vaccine strain
MA Xiao-jing1, YE Feng1,2, LIU Li-ya1, GU Wen-xi1, ZHONG Qi1, YI Xin-ping1
1. Institute of Veterinary Medicine, Xinjiang Academy of Animal Science, Urumqi 830011, China; 2. College of Animal Veterinary, Xinjiang Agriculture University, Urumqi 830052, China
Abstract：This study aimed to establish a dual-fluorescence quantitative PCR method to differentiate between the Brucella abortus A19-△VirB12 mutant vaccine strain and the wild virus infection strain. Two pairs of specific primers and two probes were designed on the basis of VirB8 from the type IV secretion system of Brucella and the VirB12 gene deletion sequence of the Brucella abortus A19-△VirB12 vaccine strain. After screening of the primers and optimization of both the reaction system and conditions, the genomic DNA of Brucella abortus A19-△VirB12 mutant vaccine strain, Brucella abortus A19 vaccine strain, Brucella melitensis M5 vaccine strain, Brucella suis S2 vaccine strain, Escherichia coli and Salmonella were amplified by real-time PCR to evaluate the specificity of the method. Plasmids positive for the VirB12 gene and VirB8 gene fragments of Brucella were constructed. After 10 fold serial dilution, real-time PCR amplification was performed to determine the sensitivity of the method. The results indicated high specificity. The VirB8 gene and VirB12 gene were simultaneously amplified in the genomic DNA of the Brucella abortus A19 vaccine strain, Brucella melitensis M5 vaccine strain and Brucella suis S2 vaccine strain. The VirB8 gene was amplified for only the Brucella abortus A19-△B12 mutant vaccine strain, whereas no amplification of Escherichia coli and Salmonella was observed. The detection limits of this method for the VirB8 gene and VirB12 gene were 102 copies/μL and 103 copies/μL respectively. In summary, a dual-fluorescence quantitative PCR method with high sensitivity and specificity was established, thus supporting the future identification of Brucella A19-△B12 mutant vaccine immunized cattle and wild virus infected cattle.
马晓菁, 叶锋, 刘丽娅, 谷文喜, 钟旗, 易新萍. 牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方-法的建立[J]. 中国人兽共患病学报, 2022, 38(7): 638-642.
MA Xiao-jing, YE Feng, LIU Li-ya, GU Wen-xi, ZHONG Qi, YI Xin-ping. Establishment of a dual real-time fluorescent PCR method for detecting the Brucella abortus A19-△VirB12 mutant vaccine strain. Chinese Journal of Zoonoses, 2022, 38(7): 638-642.
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