基于结核菌素与CFP10/ESAT6的牛IFN-γ检测法在牛结核病诊断中的比较研究  

Comparison between PPD and CFP10/ESAT6 Based Bovine IFN-γ Assays in Diagnosis of Bovine Tuberculosis

作　　者：陈颖钰[1,2] 邓铨涛[1,2] 郭爱珍[1,2] 晁彦杰[1,2] 匡有吉 于清龙[1,2] 陈焕春 

机构地区：[1]华中农业大学农业微生物学国家重点实验室 [2]华中农业大学动物医学院预防兽医学省重点实验室,武汉430070

出　　处：《中国奶牛》2008年第6期12-14,共3页China Dairy Cattle

基　　金：国家973项目(2006CB504400);国家"十一"科技攻关奶业重大专项(2006BAD04A05;2006BAD04A12);武汉市科技攻关课题(20066002056)

摘　　要：本研究通过比较三种刺激物(牛结核菌素、禽结核菌素和牛型结核菌特异性抗原CFP10/ESAT6对结核菌素皮内变态反应阳性牛的IFN-γ刺激反应,探讨IFN-γ检测法在我国牛结核病诊断中的应用前景。无菌采集22头结核菌素皮内变态反应阳性牛的血液,肝素抗凝。每1mL全血与1mLRPMI1640完全培养基混合均匀,并加入0.1mL(20μg)PHA(阳性对照孔)、0.1mL(20μg PHA)CFP10/ESAT6融合蛋白、0.1mL(2000U)牛结核菌素(PPD/B)、0.1mL(2500U)禽结核菌素(PPD/A)或等量PBS(无刺激阴性对照),37℃培养过夜。次日用夹心ELISA法检测各刺激组0.1mL培养上清的IFN-γ,以OD630表示IFN-γ浓度。结果,特异性抗原CFP10/ESAT6刺激组与牛结核菌素(PPD/B)刺激组的IFN-γ反应具有良好的相关性,相关系数为0.84。但CFP10/ESAT6刺激组IFN-γ浓度与牛和禽PPD的比较反应(以两刺激组IFN-γ浓度差值表示)间无相关性,相关系数为-0.11。分析禽PPD组的IFN-γ反应,发现实验牛中有少数牛对禽PPD有反应。以OD630=0.17为阳性反应切割值,牛PPD检出阳性牛21头,CFP10/ESAT6检出20头,牛和禽PPD比较反应(以OD值差值表示)检出14头,禽PPD阳性反应3头。扣除禽PPD阳性反应牛后,牛和禽PPD比较反应与牛PPD刺激组的相关系数增至0.54。结果表明,牛PPD的IFN-γ释放反应检测灵敏度最高。当出现牛型结核菌与环境分枝杆菌混合感染时,应用牛和禽PPD比较反应检测牛结核的准确度低,混合感染牛被误判为结核阴性牛。而基于CFP10/ESAT6的IFN-γ释放反应不受环境分枝杆菌的影响,检测具有良好的特异性与敏感性。The current research was aimed to compare the significance of three stimulators including bovine PPD, avian PPD and Mycobacterium boris specific antigen CFP10/ESAT6 in diagnosis of bovine tuberculosis. The heparinized whole blood from 22 positive cows detected with PPD skin test was collected and each lmL blood in lmL RPMI 1640 was stimulated with 0.1mL （20μg） PHA （stimulation positive control）, 0.1mL （2000U） bovine PPD, 0.1mL（250OU） avian PPD, 0.1 mL（20μg） CFP10ESAT6, or equal volume of PBS （stimulation negative control） respectively and incubated overnight at 37℃. The IFN-γ in the culture was detected with a sandwich ELISA. As a result, there was a good agreement between the groups of bovine PPD and CFP10/ESAT6 stimulation with a correlation coefficient（r） of 0.84. However, there is very low correlation between IFN-γ, release in bovine PPD group and comparative IFN-γ release resulting from the difference of bovine and avian PPD with r value of -0.24. Some of the bovine PPD reactors responded to avian PPD either. Taken OD630=0.17 as the cutoff value, there were 21 positive reactors to bovine PPD, 20 to CFP10/ ESAT6, and 3 to avian PPD. The comparative IFN-γ release detected only 14 positive cows. If the 7 avian PPD reactors with OD630 over 0.1 were excluded, the correlation between bovine PPD group and comparative IFN-γ release was increased to 0.69, and between CFP10/ESAT6 group and comparative IFN-γrelease to 0.61. In conclusion, bovine PPD based IFN-γ release assay is most sensitive, while the comparative IFN-γ release is the least. When cows are co-infected with both M. bovis and environmental mycobacteria, the positive cows with M. boris infection is likely diagnosed to be false negative by comparative IFN-γ release.In contrast, CFP10/ESAT6 based IFN-γ release assay is a promising method with high specificity and sensitivity.

关 键 词：牛结核 IFN—γ 牛结核菌素 CFP10 ESAT6 禽结核菌素 

分 类 号：S855.2[农业科学—临床兽医学]