布鲁氏菌微滴式数字PCR方法的建立与应用

目的 建立可对布鲁氏菌准确定量的微滴式数字PCR(Droplet digital PCR,ddPCR)方法。方法 使用以布鲁氏菌bscp31基因为靶标的引物和探针,摸索最佳的引物和探针工作浓度,确定反应体系和扩增条件,评价所建立方法的灵敏性、特异性及对疫情相关血液标本的检测效果。结果 ddPCR反应体系中最佳引物和探针浓度分别为0.8 μmol/L和0.4 μmol/L;最佳扩增条件:95 ℃ 10 min;95 ℃ 30 s,60 ℃ 1 min,循环40次;98 ℃酶失活10 min。布鲁氏菌核酸DNA为0.96 ng/反应~3.68 fg/反应时,ddPCR检出靶标基因拷贝数为2.00×10⁵拷贝/反应~1拷贝/反应,ddPCR测得值的对数值与DNA浓度的对数值有线性关系。在疫情相关血液标本DNA的检测中,ddPCR阳性率远高于培养法和试管凝集法;16份人血液标本中靶标基因浓度为5~65拷贝/mL血液(平均29拷贝/mL血液),5份羊血液标本中靶标基因浓度为25~245拷贝/mL血液(平均98拷贝/mL血液)。结论 ddPCR灵敏性高,可检测微量核酸DNA,适合临床血液标本的检测以及布病病原体的分子流行病学调查。

Establishment and application of droplet digital PCR in the detection of Brucella

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.