1种布鲁氏菌微滴式数字PCR检测方法的建立

A Droplet Digital PCR Method for Detection of Brucella

本研究旨在构建1种布鲁氏菌(Brucella)微滴式数字PCR方法(droplet digital PCR,ddPCR)。以布鲁氏菌BCSP 31基因为靶基因,选取保守区域设计引物和探针,构建了布鲁氏菌微滴式数字PCR方法。然后优化了ddPCR反应中的引物、探针浓度及退火温度,并进行方法的灵敏度、特异性和重复性试验。结果表明,ddPCR的最佳引物和探针浓度分别为900 nmol·L^(-1)和250 nmol·L^(-1),最佳的退火温度为58℃。ddPCR的线性良好,最低检测下限为1.12 copies·μL^(-1),与大肠杆菌O:157菌株等其他4种细菌无交叉反应,而且批内重复性和批间重复性都较好。综上表明,本研究建立的ddPCR方法灵敏度高、特异性强,可对布鲁氏菌感染的临床样品进行定量检测。

This experiment was conducted to establish a droplet digital PCR(ddPCR)method for detection of Brucella.A ddPCR method was developed,the primers and probes were designed based on the conservative regions of BCSP31 gene of Brucella.The concentration of primer and probe,the annealing temperature were optimized.The sensitivity,specificity and reproducibility of ddPCR method were evaluated.In results,the optimal primer concentration and the probe concentration were 900 nmol·L^(-1) and 250 nmol·L^(-1),the optimum annealing temperature was 58℃.The detection limit of ddPCR method was 1.12 copies·μL^(-1) with a good linear response,it had no cross reaction with E.coli O:157 and other four bacterium,both the intra-batch reproducibility and the inter-batch reproducibility were good.All the results showed that Brucella ddPCR was sensitive and specific,it was suitable for quantitative detection of clinical samples infected with Brucella.