溶血曼氏杆菌微滴式数字PCR定量检测方法的建立及应用

Developmentand Application of Droplet Digital PCR for Quantification of Menobacterium Hemolyticum

基于溶血曼氏杆菌(Mannheimia haemolytica,M.haemolytica)单拷贝基因sodA,通过优化反应条件和体系,建立了溶血曼氏杆菌ddPCR(droplet digital polymerase chain reaction,ddPCR)定量检测方法。结果显示,所建立的溶血曼氏杆菌ddPCR方法特异性好;检出限(limit of detection,LOD)和定量值(limit of quantification,LOQ)均为170 CFU/m L,3个平行检测的变异系数均小于25%,重复性良好。针对系列稀释的溶血曼氏杆菌纯菌液,ddPCR法能够实现对溶血曼氏杆菌的准确定量,与平板计数结果偏差小于4%。运用所建立的ddPCR方法对15份牛鼻拭子样品进行检测,结果在10份样品中检出溶血曼氏杆菌,含量在3.07×10^(2)~9.52×10^(4)CFU/mL之间。本研究所建立的ddPCR方法适用于临床鼻拭子样品中溶血曼氏杆菌的精准定量分析,可为牛群中溶血曼氏杆菌的监测和治疗提供有效的技术支持。

Based on the single copy gene sodA of M.haemolyticus,a ddPCR assay for the quantitative detection of M.haemolyticus was developed after the optimization of reaction condition and system.The results showed that the developed M.haemolytica ddPCR assay had good specificity.Both of the LOD and LOQ were 170 CFU/mL,and the coefficient of variation of three parallel tests were less than 25%,which demonstrated good repeatability.For the gradient diluted pure culture solution of M.haemolyticus,the accurate quantification of M.haemolyticus could be realized by the developed ddPCR assay,and the deviation compared with plate counting was less than 4%.Among the 15 cow nose swab samples,M.haemolyticus DNA was detected in 10samples by the developed ddPCR assay with the content calculated as 3.07×10^(2)~9.52×10^(4) CFU/mL.The developed ddPCR assay was suitable for the accurate quantitative analysis of M.haemolytica in clinical swab samples,and can provide effective technical supports for the monitoring and treatment of M.haemolytica infection in cattle.