[1]卢春霞,刘长彬,万鹏程,等.绵羊ovPAG7间接竞争酶联适配体检测方法的建立与应用[J].江苏农业学报,2022,38(03):730-738.[doi:doi:10.3969/j.issn.1000-4440.2022.03.019]
　LU Chun-xia,LIU Chang-bin,WAN Peng-cheng,et al.Establishment and application of indirect competitive enzyme-linked aptamer assay for ovPAG7 in sheeps[J].,2022,38(03):730-738.[doi:doi:10.3969/j.issn.1000-4440.2022.03.019]
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绵羊ovPAG7间接竞争酶联适配体检测方法的建立与应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
38
期数:
2022年03期
页码:
730-738
栏目:
畜牧兽医·水产养殖
出版日期:
2022-06-30

文章信息/Info

Title:
Establishment and application of indirect competitive enzyme-linked aptamer assay for ovPAG7 in sheeps
作者:
卢春霞1; 2; 刘长彬2; 万鹏程2; 郭延华2; 陈霞3; 李红敏3; 卢守亮2
(1.长江师范学院现代农业与生物工程学院，重庆408100；2.新疆农垦科学院省部共建绵羊遗传改良与健康养殖国家重点实验室，新疆石河子832000;3.新疆农垦科学院分析测试中心，新疆石河子832000）
Author(s):
LU Chun-xia1; 2; LIU Chang-bin2; WAN Peng-cheng2; GUO Yan-hua2; CHEN Xia3; LI Hong-min3; LU Shou-liang2
(1.School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China;2.State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China;3.Analysis and Testing Center of Xinjiang Academy of Agriculture and Reclamation Science, Shihezi 832000, China)
关键词:
妊娠相关糖蛋白; 适配体; 早期妊娠诊断; 绵羊
Keywords:
pregnancy associated glycoprotein; aptamer; early pregnancy diagnosis; sheep
分类号:
S826.3+5
DOI:
doi:10.3969/j.issn.1000-4440.2022.03.019
文献标志码:
A
摘要:
为建立一种简便、经济、无创的应用于绵羊早期妊娠诊断的快速检测技术，以绵羊妊娠相关糖蛋白7（Ovine pregnancy-associated glycoproteins-7, ovPAG7）核酸适配体为分子识别探针，建立一种间接竞争酶联适配体（Indirect competitive enzyme-linked aptamer assay, ic-ELAA）检测ovPAG7的新方法，并对ovPAG7包被质量浓度、包被缓冲液、封闭条件、适配体浓度、链霉亲和素-辣根过氧化物酶（SA-HRP）稀释比例等检测条件进行优化，将建立的方法应用于人工授精后28 d的中国美利奴羊血清检测，检测结果与妊娠相关糖蛋白-酶联免疫吸附测定（PAG-ELISA）商业化试剂盒和B超检测法进行比较。结果显示，在ovPAG7包被质量浓度为2.0 μg/ml、包被缓冲液为50 mmol/L碳酸盐缓冲液（pH 9.6）、1.0%牛血清蛋白质封闭2 h、适配体浓度为40 nmol/L、SA-HRP稀释比例为1∶40 000 (体积比)、室温下竞争反应时间为40 min等优化条件下，ovPAG7在缓冲液体系和血清体系中质量浓度的线性范围均为0.1～50.0 ng/ml (R2>0.994) ，半数抑制质量浓度（IC50）分别为1.40 ng/ml和2.11 ng/ml，检测限分别为0.11 ng/ml和0.19 ng/ml。ovPAG7在空白样品中加标回收率为96.4%～106.8%，相对标准偏差小于6.0%。妊娠诊断结果显示，ic-ELAA法的诊断敏感性、特异性和准确率分别为96.0%、83.3%和91.2%，与B超检测结果一致性较高（Kappa值=0.810）。
Abstract:
The aim of the experiment was to establish a rapid and new detection technique that can be applied simply, economically and noninvasively in diagnosis of sheeps in early period of pregnancy. An indirect competitive enzyme-linked aptamer assay (ic-ELAA) was developed using ovine pregnancy-associated glycoproteins-7 (ovPAG7) nucleic acid aptamer as molecular probe for the detection of ovPAG7. Several detecting conditions including mass concentration of coated ovPAG7, coating buffer, blocking conditions, concentration of aptamer and dilution ratio of streptavidin-horseradish peroxidase (SA-HRP) were optimized. Subsequently, the serum samples from Chinese Merino ewes 28 days after insemination were analyzed by applying the ic-ELAA method proposed. The test results were compared with ultrasound B detection method and PAG-ELISA (pregnancy associated glycoproteins-enzyme linked immunosorbent assay) kit. The results demonstrated that, the optimized conditions were 2.0 μg/ml mass concentration of coated ovPAG7, 50 mmol/L carbonate with pH 9.6 as coating buffer, blocking the wells with 1.0% bovine serum albumin for 2 h, the aptamer concentration was 40 nmol/L, the dilution ratio (in volume) of SA-HRP was 1∶40 000, the competitive reaction time at room temperature was 40 min. Under the optimal conditions, the linear range of mass concentration of ovPAG7 in both buffer system and serum system was 0.1-50.0 ng/ml (R2>0.994), with median inhibition concentration (IC50) of 1.40 ng/ml and 2.11 ng/ml respectively, and the limit of detection were 0.11 ng/ml and 0.19 ng/ml respectively. The recovery rate of ovPAG7 in blank samples was between 96.4% and 106.8%, and the relative standard deviations were all less than 6.0%. Results of pregnancy diagnosis showed that, the diagnostic sensitivity, specificity and accuracy of ic-ELAA method were 96.0%, 83.3% and 91.2% respectively, which showed high consistency with detection results by ultrasound B method (Kappa=0.810).

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备注/Memo

备注/Memo:
收稿日期：2021-07-25基金项目：新疆生产建设兵团科技创新人才计划项目(2020CB022)；重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0080)；国家重点实验室优秀中青年人才培养引导计划专项（SKLSGIH2017A02）作者简介：卢春霞（1978-），女，河南商丘人，博士，教授，主要从事快速检测技术研究。(E-mail)shzlcx2002@163.com通讯作者：刘长彬，(E-mail)xlchangbin@163.com
更新日期/Last Update: 2022-07-07