Abstract：
The pregnancy-associated glycoproteins (PAGs) have been widely used as biomarkers for the early diagnosis of pregnancy in cattle and sheep. This study aimed to obtain the single-stranded DNA aptamers that specifically bound to ovine pregnancy-associated glycoprotein 7 (ovPAG7) with high affinity (Kd = 9.8-32.4 nM) using real serum sample-assisted FluMag-systematic evolution of ligands by exponential enrichment (SELEX). Subsequently, the selected aptamers were applied to fabricate an ultrasensitive colorimetric aptasensor for ovPAG7 detection based on functionalized magnetic microparticles and hybridization chain reaction. Under the optimized conditions, the colorimetric aptasensor exhibited a broad linear range (0.2-500 ng mL-1), low detection limit (0.081 ng mL-1), good recovery rate (94.5-109.1%), and high repeatability (relative standard deviation of 4.02-8.16%) in ovPAG7-spiked serum. Furthermore, this aptasensor was applied to measure the ovPAG7 in serum samples of ewes for pregnancy diagnosis. Blood samples were collected from Chinese Merino ewes on days 22, 28 after artificial insemination (AI) for ovPAG7 detection, respectively. Transrectal ultrasonography diagnosis of pregnancy 45 days after AI was the reference (gold) standard for all PAG tests. Diagnostic sensitivity, specificity, and accuracy of the proposed aptasensor were 95.8, 87.5, and 92.5% at day 22 and 95.8, 90.6, and 93.7% at day 28, respectively. The degree of agreement (Kappa) between developed aptasensor and ultrasonography diagnosis 22 and 28 day after AI were higher than 0.8. These results illustrated that the aptasensor was proved to be a sensitive, reliable and cost-effective way of measuring PAG and might be a useful means of pregnancy detection in ewes.
摘要：
妊娠相关糖蛋白(PAGs)已被广泛用作牛和羊妊娠早期诊断的生物标志物。本研究旨在通过指数富集(SELEX),使用真实血清样品辅助的FluMag系统进化配体,获得以高亲和力(Kd=9.8-32.4nM)特异性结合绵羊妊娠相关糖蛋白7(ovPAG7)的单链DNA适体。随后，选择的适体被用于制造超灵敏的比色aptasensor,用于基于功能化磁性微粒和杂交链反应的ovPAG7检测.在优化条件下,比色传感器表现出广泛的线性范围（0.2-500ngmL-1），低检测限（0.081ngmL-1），良好的回收率（94.5-109.1%），并且在ovPAG7加标血清中具有较高的重复性（相对标准偏差为4.02-8.16％）。此外，该aptasensor用于测量母羊血清样本中的ovPAG7，以进行妊娠诊断。在人工授精(AI)后第22、28天从中国美利奴羊母羊采集血样进行ovPAG7检测,分别。AI后45天经直肠超声检查诊断妊娠是所有PAG测试的参考（金）标准。诊断灵敏度,特异性，建议的aptasensor的准确度在第22天分别为95.8、87.5和92.5％，在第28天分别为95.8、90.6和93.7％。AI后22天和28天，发育的aptasensor与超声诊断之间的一致度（Kappa）高于0.8。这些结果表明，aptasensor被证明是一个敏感的，测量PAG的可靠且具有成本效益的方法，可能是母羊妊娠检测的有用手段。
