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中国农学通报

中国农学通报 ›› 2023, Vol. 39 ›› Issue (2): 75-81.doi: 10.11924/j.issn.1000-6850.casb2021-0985

所属专题： 畜牧兽医

• 畜牧·动物医学·蚕·蜂 • 上一篇    下一篇

牛妊娠相关糖蛋白6快速检测试纸条的制备及应用

李志明1(), 许海涛2, 高玉平3, 蔡德霖1, 王相国1, 肖龙菲1, 安红1, 王颖秋1, 王骞1, 郭勇1, 齐晓龙1, 倪和民1()   

  1. 1 北京农学院动物科学技术学院，北京 102206
    2 全国畜牧总站，北京 100125
    3 天津市蓟州区下仓镇人民政府，天津 301905
  • 收稿日期:2021-10-18 修回日期:2022-09-13 出版日期:2023-01-15 发布日期:2023-01-17
  • 作者简介:

    李志明，男，1996年出生，湖南安化人，硕士，主要从事动物生殖机理调控与产科疾病方面的研究。通信地址：102206 北京农学院动物科学技术学院，E-mail：。

  • 基金资助:
    北京市科协北京金桥种子工程项目“人工输精奶牛早期妊娠血液诊断方法的建立”(ZZ19002); 北京市农业农村局项目“北京市奶牛产业创新团队岗位专家项目”(BAIC05-2017)

Preparation and Application of Test Strip for Rapid Detection of Bovine Pregnancy-associated Glycoprotein 6

LI Zhiming1(), XU Haitao2, GAO Yuping3, CAI Delin1, WANG Xiangguo1, XIAO Longfei1, AN Hong1, WANG Yingqiu1, WANG Qian1, GUO Yong1, QI Xiaolong1, NI Hemin1()   

  1. 1 College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206
    2 National Animal Husbandry Services, Beijing 100125
    3 People’s Government of Xiacang Town, Jizhou District, Tianjin 301905
  • Received:2021-10-18 Revised:2022-09-13 Online:2023-01-15 Published:2023-01-17

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摘要：

奶牛妊娠的早期诊断至关重要，妊娠相关糖蛋白6(PAG6)是母体胎盘组织分泌至外周循环中的特异蛋白质，因此，本研究致力于制备牛妊娠相关糖蛋白6快速检测试纸条。制备牛妊娠相关糖蛋白6多克隆抗体，将其稀释后加样至NC膜上，通过固定和封闭制备出一种可检测boPAG6的快速检测试纸条，在试纸条上加入待检血清进行孵育后洗涤，再加入二抗孵育后洗涤，最后加入显色液显色后进行快速检测。标准化快速检测试纸条是将抗体用PBS稀释成10 μg/mL点样至NC膜上，在37℃温箱中固定抗体30 min，经5%的脱脂奶粉封闭后制成。待检血清滴加至试纸条后，在37℃温箱中孵育1 h后，进行洗涤和二抗孵育，最后在DAB显色试剂盒作用下显色3 min即可判定。临床检测的初步应用表明，在未孕与怀孕奶牛的混合血清中仍然可以判定是否怀孕，快速检测试纸条特异性强；血清在稀释3200倍的最低检测限时，快速检测试纸条的敏感性良好；而且该结果与ELISA的符合率在80%之上。试验重复性良好，并能在4℃和-20℃条件下保存2周。建立了一种奶牛早期妊娠诊断的快速检测试纸条并应用于初步的妊娠诊断。

关键词: 奶牛, 早期妊娠, 诊断, 妊娠相关糖蛋白6, 快速检测试纸条

Abstract:

The early diagnosis of bovine pregnancy is very important. Pregnancy-associated glycoprotein 6 (PAG6) is a specific protein secreted by maternal placental tissue to the peripheral circulation. Therefore, this study is to prepare a test strip for rapid detection of bovine pregnancy-associated glycoprotein 6. The polyclonal antibody against bovine pregnancy-associated glycoprotein 6 was prepared and diluted and added to the NC membrane. A rapid detection strip for boPAG6 was prepared by fixing and sealing. The serum to be tested was added to the test strip for incubation and then washed, and then the second antibody was added for washing. Finally, the chromogenic solution was added for rapid detection. The standardized rapid test strip diluted the antibody to 10 μg/mL with PBS and put it on the NC film. The antibody was fixed at 37℃ for 30 min and sealed with 5% skim milk powder. The serum to be tested was dropped onto the test strip and incubated at 37℃ for 1 h, then washed and secondary antibody was incubated. Finally, DAB chromogenic kit was used for 3 min to determine the detection. The preliminary application in clinical detection showed that pregnancy could still be determined in the mixed serum of non-pregnant cows and pregnant cows, and the specificity of rapid detection of the test strip was strong. The sensitivity of the rapid detection of the test strip was good at the minimum detection limit of 3200 times dilution of serum. The coincidence rate with ELISA was above 80%. The experiment has good repeatability and the test strip can be stored at 4℃ and -20℃ for 2 weeks. The test strip for rapid detection of early pregnancy diagnosis in dairy cows is established and can be applied to preliminary pregnancy diagnosis.

Key words: dairy cows, early pregnancy, diagnosis, pregnancy-associated glycoprotein 6, test strip for rapid detection