奶牛乳腺上皮细胞中bta-miR-29b实时荧光定量PCR检测方法的建立与应用  

Establishment and application of RT-qPCR for detection of bta-miR-29b in bovine mammary epithelial cells

作　　者：潘晓乐 扈登强 穆爱娟 马继贤 周学章[1] 韩博[4] 王东[1] PAN Xiao-le;HU Deng-qiang;MU Ai-juan;MA Ji-xian;ZHOU Xue-zhang;HAN Bo;WANG Dong(School of Life Science,Ningxia University,Yinchuan 750021,China;Ningxia Hongfeng Technical Service Co.Ltd.,Yinchuan 750021,China;Agriculture and Rural Affairs Bureau of Helan County,Helan 760200,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]宁夏大学生命科学学院,宁夏银川750021 [2]宁夏弘丰技术服务有限公司,宁夏银川750021 [3]宁夏贺兰县农业农村局,宁夏贺兰750200 [4]中国农业大学动物医学院,北京100193

出　　处：《中国兽医科学》2021年第11期1369-1375,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31760751);宁夏重点研发计划项目(2019BBF02027)。

摘　　要：为了检测金黄色葡萄球菌(S.aureus)感染的奶牛乳腺上皮细胞(bMECs)中bta-miR-29b表达,本研究建立bta-miR-29b实时荧光定量PCR(RT-qPCR)方法,并对该方法的特异性、敏感性和重复性进行评估,利用该方法检测金黄色葡萄球菌感染bMECs中bta-miR-29b的表达水平。结果显示,构建的RT-qPCR方法在bta-miR-29b标准品10 pg/20μL~1×10^(-4)pg/20μL浓度范围内,初始模板量和Ct值之间呈现良好的线性关系,线性相关系数R^(2)=0.9997。特异性试验结果显示,对bta-miR-29b标准品样本检测结果为阳性,对牛大肠杆菌和链球菌样本检测结果均为阴性,说明该方法特异性强;敏感性试验结果显示,对bta-miR-29b标准品的最低检测模板浓度为1×10^(-4)pg/20μL,比常规PCR检测的敏感性高100倍,表明该方法敏感性高;重复性试验结果显示,组内和组间的变异系数均小于1%,说明该方法重复性好。临床样本的检测结果显示,金黄色葡萄球菌感染bMECs后第24小时bta-miR-29b的表达量极显著下调(P<0.01)。上述结果表明,本研究建立的RT-qPCR方法特异性强、敏感性高、重复性好,能够准确检测金黄色葡萄球菌感染bMECs中bta-miR-29b的表达,为今后bta-miR-29b的表达分析及其生物学功能的研究提供了研究手段。In order to quantitatively detect bta-miR-29 b in bovine mammary epithelial cells(bMECs)challenged with Staphylococcus aureus(S.aureus),a real-time reverse transcription quantitative PCR(RT-qPCR)assay was developed and its specificity,sensitivity and reproducibility were evaluated,respectively in this study.This protocol was also applied to detect bta-miR-29 b in bMECs infected with S.aureus.Results showed that there was a good linear relationship between the initial template amount and Ct value in the template concentration of bta-miR-29 b standard range of 10 pg/20μL to 1×10^(-4) pg/20μL,with the linear correlation coefficient up to 0.9997.The bta-miR-29 b standard sample had a positive result,but bovine E.coli and Streptococcus spp.could not be detected in RT-qPCR,indicating RT-qPCR developed had a high specificity.The minimum template concentration of RT-qPCR was 1×10^(-4) pg/20μL,which was lower 100-fold than that of conventional PCR,showing a high sensitivity.The coefficients of variation of RT-qPCR was less than 1%in the reproducibility test,suggesting a good reproducibility.The expression of bta-miR-29 b in bMECs infected with S.aureus at 24 h was significantly down-regulated(P<0.01)using RT-qPCR.This RT-qPCR developed in this study not only has high specificity,high sensitivity,and good reproducibility,but also can accurately detect the expression of bta-miR-29 b in b MECs infected with S.aureus,which provides a research tool for further analyzing the expression of bta-miR-29 b and verifying its biological function.

关 键 词：实时荧光定量PCR 金黄色葡萄球菌 奶牛乳腺上皮细胞 bta-miR-29b 

分 类 号：S852.611[农业科学—基础兽医学]