牛结核γ-干扰素夹心ELISA检测试剂盒的研制与评价  

Development and evaluation of gamma-interferon sandwich ELISA kit in herds infected with Mycobacteriumbovis

作　　者：李昕 徐正中 单法 夏爱鸿 孟闯 沈也驰 陈义平[3] 南文龙[3] 陈祥 焦新安 

机构地区：[1]扬州大学,江苏省人兽共患病学重点实验室/江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [2]扬州大学,农业部农产品质量安全生物性危害因子(动物源)控制重点实验室,扬州225009 [3]中国动物卫生与流行病学中心,青岛266032

出　　处：《中国人兽共患病学报》2017年第12期1060-1065,共6页Chinese Journal of Zoonoses

基　　金：国家重点研发计划(No.2017YFD0500300);国家自然科学基金(No.31602031);江苏科技计划项目(No.BE2015343,No.BK20160466,No.BK20171285,No.16KJB230003);江苏‘青蓝工程’‘六大人才高峰’(No.SWYY-083);优势学科建设工程项目联合资助~~

摘　　要：目的研制一种牛结核γ-干扰素夹心ELISA检测试剂盒,并在奶牛结核病检疫中进行初步应用和评价。方法建立牛结核γ-干扰素夹心ELISA检测方法,基于此法试制3批试剂盒,并对试剂盒的灵敏度、可重复性和保存期进行评价,其后进行初步应用,对961头奶牛采用单纯颈部皮试变态反应、商品化BOVIGAMTM试剂盒与试制的牛结核γ-干扰素夹心ELISA检测试剂盒进行同步检测。结果该试剂盒对质控样品的最低检出量达到8.21ng/mL,而且检测重复性良好,试剂盒保存期达12个月以上。在进行临床试验时,试制的试剂盒与单纯颈部皮试变态反应相比,阳性一致率为70.59%,阴性一致率为99.20%,总一致率为98.44%;与BOVIGAMTM试剂盒相比,阳性一致率为91.30%,阴性一致率为99.78%,总一致率为99.58%。其灵敏度为85.00%,特异性为100.00%。结论成功建立牛结核γ-干扰素夹心ELISA检测方法,并研制出相应试剂盒,具有良好应用前景。This study was aimed to develop a sandwich ELISA kit for the diagnosis of bovine tuberculosis.And it was applied and evaluated in the quarantine of bovine tuberculosis.We established a bovine IFN-γrelease method in vitro and developing three batches of kits.The sensitivity,repeatability and retention period of the kit were all evaluated.Totally 961 serum samples were tested using the developed sandwich ELISA kit tuberculin skin test and a commercial ELISA kit.Our results showed that the detection limit of this ELISA was 8.21 mg/mL.The repeatability tests showed good reproducibility in the intra-assay and inter-assay.At the same time,the retention period of the kit was more than 12 months.Compared with the tuberculin skin test,the positive coincidence rate was 70.59% and the negative coincidence rate was 99.20%,while the total coincidence rate was 98.44%.And compared with the BOVIGAMTMkit,the positive coincidence rate was 91.30% and the negative coincidence rate was 99.78%,while the total coincidence rate reached 99.58%.At the same time,the sensitivity and specificity of the sandwich kit were 85.00% and100%,respectively.We established a bovine IFN-γrelease method in vitro and developing corresponding kits successfully have a good application prospect.

关 键 词：牛结核病 Γ-干扰素 夹心ELISA 试剂盒 敏感性 特异性 

分 类 号：R378.91[医药卫生—病原生物学]