Enzyme-linked Aptamer Assay for Visual Detection of bPAG9 and Its Application for Pregnancy Diagnosis in Dairy Cows (Bos taurus)
LU Chun-Xia1, LIU Chang-Bin2,*, ZOU Ping2, LIN Xiang-Qun3
1 School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China; 2 China Key Laboratories of Sheep Breeding and Reproduce/Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China; 3 Xinjiang Shihezi Vocational Technical College, Shihezi 832000, China
Abstract：Pregnancy-associated glycoproteins (PAGs) immunoassay have been widely used for early pregnancy diagnosis in livestock. The use of an antibody as a probe for detection has some drawbacks of long preparation period and high cost. To enrich the diagnosis techniques of early pregnancy in dairy cows (Bos taurus) and reduce detection cost, a enzyme-linked aptamer assay (ELAA) was developed for visual detection of bovine pregnancy-associated glycoprotein 9 (bPAG9) based on sandwich-type format strategy. Several experimental conditions had been investigated, including the concentration of aptamer, horseradish peroxidase (HRP)-aptamer conjugates and blocking agent, incubation time, and coating buffer. Under the optimal conditions, the calibration curves for bPAG9 showed good linearities in the range of 0.5~100 ng/mL with correlation coefficients (R) better than 0.998. The detection limit of 0.22 ng/mL by the instrument, and the limit of visual detection was found to be 0.5 ng/mL by naked-eye observation. The recovery experiment was performed by spiking blank samples with 1, 10 and 100 ng of bPAG9. The average recoveries of bPAG9 were in the range of 93.2%~105.8%, with relative standard deviations of 3.51%~7.13%. Subsequently, the 60 serum samples from pregnant and nonpregnant cows at 30 d after insemination were analyzed by the proposed ELAA. The test results were confirmed with ultrasonography. The diagnostic accurate rate of ELAA was 91.7%. This suggested that the proposed method had good accuracy and reliability in real samples. The enzyme-linked aptamer assay was shown to provide a simple, economical, reliable rapid detection technology for pregnancy diagnosis in dairy cows.
卢春霞, 刘长彬, 周平, 林祥群. 酶联适配体可视化检测牛妊娠相关糖蛋白9 (bPAG9)及其在奶牛早孕检测中的应用[J]. 农业生物技术学报, 2021, 29(7): 1407-1416.
LU Chun-Xia, LIU Chang-Bin, ZOU Ping, LIN Xiang-Qun. Enzyme-linked Aptamer Assay for Visual Detection of bPAG9 and Its Application for Pregnancy Diagnosis in Dairy Cows (Bos taurus). 农业生物技术学报, 2021, 29(7): 1407-1416.
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