一种检测奶样中布鲁氏菌抗体免疫层析方法的建立
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摘要
通过制备脂多糖(LPS)并将其作为检测抗原,建立了一种检测奶样中布鲁氏菌抗体的酶联免疫层析方法。通过对处理方法和反应条件的优化,制备出敏感性高、特异性强的布鲁氏菌抗体检测试纸条。该试纸条敏感性为98.0%,抗体滴度为1:2~1:8,特异性为96.0%,且与其他常见病原无交叉反应,批内、批间重复性良好。保存期加速试验显示,该试纸条在2~30℃下可保存12个月;符合率验证显示,该方法与标准奶样的符合率达94.9%。上述结果表明,该检测方法具有敏感性高、特异性强以及方便、快捷等优点,适用于现场大批量检测,因而可应用于奶牛场布鲁氏菌病的快速诊断。
In this paper,the immunochromatography assay(ICA)for detection of Brucella antibodies in milk samples was established by preparing lipopolysaccharide(LPS)as a detection antigen. By optimizing processing method and reaction conditions,the Brucella antibodies test kit with strong sensitivity and speci?city was established.Results showed that the sensitivity of the kit was 98.0%,the antibody titer was 1:2 to 1:8,and the specificity was96.0%. No any cross-reaction was observed between the kits with other common pathogens,besides,the repeatability of intra and inter assay was good. The accelerated shelf life test(ASLT)showed that the kit could be stored for 12 months at 2 ℃ to 30 ℃;and the coincidence rate between the method and standard milk samples was veri?ed to be94.9%. It was concluded that the method,with the advantages of strong sensitivity and speci?city,convenience and rapidity,was suitable for mass detection in the ?eld,and could be used for rapid diagnosis of brucellosis in dairy farms.
In this paper,the immunochromatography assay(ICA)for detection of Brucella antibodies in milk samples was established by preparing lipopolysaccharide(LPS)as a detection antigen. By optimizing processing method and reaction conditions,the Brucella antibodies test kit with strong sensitivity and speci?city was established.Results showed that the sensitivity of the kit was 98.0%,the antibody titer was 1:2 to 1:8,and the specificity was96.0%. No any cross-reaction was observed between the kits with other common pathogens,besides,the repeatability of intra and inter assay was good. The accelerated shelf life test(ASLT)showed that the kit could be stored for 12 months at 2 ℃ to 30 ℃;and the coincidence rate between the method and standard milk samples was veri?ed to be94.9%. It was concluded that the method,with the advantages of strong sensitivity and speci?city,convenience and rapidity,was suitable for mass detection in the ?eld,and could be used for rapid diagnosis of brucellosis in dairy farms.
引文
[1]尚德秋.谈谈布鲁氏菌病病原学的某些问题[J].中级医刊,1979(2):9-12.
[2]姜顺求.布鲁氏菌病防治手册[J].北京:人民卫生出版社,1984.
[3]崔步云.关注中国布鲁杆菌病疫情发展和疫苗研究[J].中国地方病学杂志,2012,31(4):355-356.
[4]尚德秋,于恩庶,赵恒云.布鲁氏菌病实验诊断的非特异性反应及其鉴别[M].北京:海洋出版社,1995.
[5]中国疾病预防控制中心公共卫生监测和信息服务中心.2014年12月中国甲乙丙类传染病疫情动态简介[J].疾病监测,2015,30(1):1.
[6]ISLAM M A,KHATUN M M,WERRE S R,et al.A review of Brucella seroprevalence among humans and animals in Bangladesh with special emphasis on epidemiology,risk factors and control opportunities[J].Veterinary microbiology,2013,166(3):317-326.
[7]任燕斌.布鲁氏菌竞争ELISA检测方法的建立及应用[D].重庆:重庆理工大学,2016.
[8]左玉枉,王增利.牛布鲁氏菌BP26间接ELISA检测方法的建立[J].中国预防兽医学报,2014,36(3):223-226.
[9]王志明,赵妮.抗布鲁氏菌单克隆抗体的研制及cELISA方法的建立[J].中国兽医科学,2014,44(9):909-913.
[10]王静,胡孔新,李伟,等.2种免疫层析技术用于4种传染病及其病原体检测的研究[J].中国国境卫生检疫杂志,2006,34(31):27-34.
[11]庄金秋,梅建国,沈志强.免疫胶体金快速诊断技术在兽医临床诊断中的应用进展[J].中国动物检疫,2012,29(9):70-75.
[12]张付贤,李晓艳.鉴别布鲁菌病自然感染动物与疫苗免疫动物胶体金检测试纸条的研制[J].中国畜牧兽医,2009,36(7):79-82.
[13]邵丰尧,朱匡纪,朱明东.胶体金免疫层析法在布鲁氏菌病监测地区的现场应用[J].疾病监测,2009,24(10):753-754.
[14]严华,申厚凤.胶体金免疫层析技术的应用与展望[J].微生物学免疫学进展,2005(3):86-89.
[15]程婷婷,石峰,陈创夫,等.布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立[J].中国病原生物学杂志,2014,9(2):122-126.
[16]檀华,何君,原丽,等.布鲁氏菌病快速诊断试纸(dipstick)的研制及初步应用[J].中国人畜共患病学报,2004,20(8):718.
[17]徐卫民,朱明东,王衡,等.快速检测布鲁氏菌抗体的滴金免疫测定法的建立及应用[J].中国卫生检验杂志,2006,16(1):17-18.
[18]朱明东,杨蓉,洪林娣,等.快速诊断布鲁氏菌病胶体金免疫层析法的建立[J].中国卫生检验杂志,2008,18(7):1344-1345.
[2]姜顺求.布鲁氏菌病防治手册[J].北京:人民卫生出版社,1984.
[3]崔步云.关注中国布鲁杆菌病疫情发展和疫苗研究[J].中国地方病学杂志,2012,31(4):355-356.
[4]尚德秋,于恩庶,赵恒云.布鲁氏菌病实验诊断的非特异性反应及其鉴别[M].北京:海洋出版社,1995.
[5]中国疾病预防控制中心公共卫生监测和信息服务中心.2014年12月中国甲乙丙类传染病疫情动态简介[J].疾病监测,2015,30(1):1.
[6]ISLAM M A,KHATUN M M,WERRE S R,et al.A review of Brucella seroprevalence among humans and animals in Bangladesh with special emphasis on epidemiology,risk factors and control opportunities[J].Veterinary microbiology,2013,166(3):317-326.
[7]任燕斌.布鲁氏菌竞争ELISA检测方法的建立及应用[D].重庆:重庆理工大学,2016.
[8]左玉枉,王增利.牛布鲁氏菌BP26间接ELISA检测方法的建立[J].中国预防兽医学报,2014,36(3):223-226.
[9]王志明,赵妮.抗布鲁氏菌单克隆抗体的研制及cELISA方法的建立[J].中国兽医科学,2014,44(9):909-913.
[10]王静,胡孔新,李伟,等.2种免疫层析技术用于4种传染病及其病原体检测的研究[J].中国国境卫生检疫杂志,2006,34(31):27-34.
[11]庄金秋,梅建国,沈志强.免疫胶体金快速诊断技术在兽医临床诊断中的应用进展[J].中国动物检疫,2012,29(9):70-75.
[12]张付贤,李晓艳.鉴别布鲁菌病自然感染动物与疫苗免疫动物胶体金检测试纸条的研制[J].中国畜牧兽医,2009,36(7):79-82.
[13]邵丰尧,朱匡纪,朱明东.胶体金免疫层析法在布鲁氏菌病监测地区的现场应用[J].疾病监测,2009,24(10):753-754.
[14]严华,申厚凤.胶体金免疫层析技术的应用与展望[J].微生物学免疫学进展,2005(3):86-89.
[15]程婷婷,石峰,陈创夫,等.布鲁氏菌抗体胶体金免疫层析法快速检测技术的建立[J].中国病原生物学杂志,2014,9(2):122-126.
[16]檀华,何君,原丽,等.布鲁氏菌病快速诊断试纸(dipstick)的研制及初步应用[J].中国人畜共患病学报,2004,20(8):718.
[17]徐卫民,朱明东,王衡,等.快速检测布鲁氏菌抗体的滴金免疫测定法的建立及应用[J].中国卫生检验杂志,2006,16(1):17-18.
[18]朱明东,杨蓉,洪林娣,等.快速诊断布鲁氏菌病胶体金免疫层析法的建立[J].中国卫生检验杂志,2008,18(7):1344-1345.