Abstract：A droplet digital PCR (ddPCR) method was established for the accurate quantification of Brucella. The ddPCR method used primers and a probe targeting the Brucella bscp31 gene. The optimal reaction system and amplification conditions were explored. The analytical sensitivity and specificity of the established method and its application in the detection of epidemic-related blood samples were evaluated. The optimal concentrations of the primers and the probe were 0.8 μmol/L and 0.4 μmol/L, respectively. The optimal cycling conditions were 95 ℃ for 10 min; 40 cycles of 95 ℃ for 30 sec and 60 ℃ for 1 min; and 98 ℃ for 10 min to inactivate the enzyme. When the template Brucella DNA was 0.96 ng/reaction to 3.68 fg/reaction, the target gene was present in 2.00×105 copies/reaction to 1 copy/reaction, as shown by ddPCR. The logarithm of the copy number obtained by ddPCR had a linear relationship with the logarithm of the amount of DNA. In the detection of DNA in epidemic-related blood samples, the positive rate of ddPCR was much higher than those detected with the culture method and tube agglutination method; the target concentration in 16 human blood samples was 5-65 copies/ml blood (average 29 copies/ml blood), and the target concentration in five sheep blood samples was 25-245 copies/ml blood (average 98 copies/ml blood). The ddPCR had high sensitivity and was able to detect trace Brucella DNA. It is suitable for detection in clinical blood samples and molecular epidemiological investigation of brucellosis.
田国忠, 姜海, 薛盼盼, 朴东日, 赵鸿雁, 杨晓雯, 张京云. 布鲁氏菌微滴式数字PCR方法的建立与应用[J]. 中国人兽共患病学报, 2021, 37(4): 301-305.
TIAN Guo-zhong, JIANG Hai, XUE Pan-pan, PIAO Dong-ri, ZHAO Hong-yan, YANG Xiao-wen, ZHANG Jing-yun. Establishment and application of droplet digital PCR in the detection of Brucella. Chinese Journal of Zoonoses, 2021, 37(4): 301-305.
2021, Vol. 37 
