马Toll样受体表达水平SYBR GreenⅠ荧光定量RT-PCR检测方法的建立

畜牧兽医学报

马Toll样受体表达水平SYBR GreenⅠ荧光定量RT-PCR检测方法的建立

赵一萍，白东义，李蓓，黄金龙，张宇宏，芒来^*

（内蒙古农业大学动物科学学院，呼和浩特 010018）

收稿日期:2012-05-04 出版日期:2013-02-23 发布日期:2013-02-23
通讯作者: 芒来，博士生导师，教授，主要从事分子数量遗传学与马科学研究，E-mail：dmanglai@yahoo.com.cn
作者简介:赵一萍（1984-），女，满族，河北高碑店人，博士生，主要从事分子数量遗传学与马科学研究，E-mail：zhaoyiping_84@163.com
基金资助:
内蒙古自治区科技厅重大项目（20091404）；内蒙古农业大学科技创新团队支持计划（NDTD2010-12）

Establishment of a Real-time RT-PCR Assay Based on SYBR Green Ⅰ for Detection of the Expression of Horse Toll-like Receptor Genes

ZHAO Yi-ping, BAI Dong-yi, LI Bei, HUANG Jin-long, ZHANG Yu-hong,DUGARJAVIIN Mang-lai^*

(College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China)

Received:2012-05-04 Online:2013-02-23 Published:2013-02-23

摘要/Abstract

摘要：

旨在建立一种检测马Toll样受体（TLRs）mRNA表达水平的SYBR Green Ⅰ荧光定量RT-PCR（Rverse Transcription-Polymerase Chain Reaction）方法。参照GenBank中马TLRs的基因序列保守区设计特异性引物，以马β肌动蛋白（β-actin）mRNA为内参，建立荧光定量RT-PCR方法。结果，扩增曲线在1×10²～1×10⁸ copies·μL^-1范围内有很好的线性关系，扩增相关系数在0.990以上，扩增效率在1.0左右。熔解曲线分析表明，产物为特异单峰，无引物二聚体，具有较高的特异性和灵敏度。重复性结果表明，该方法的组内、组间变异系数均小于3.50%，重复性较好。临床样品检测结果表明，TLR1、TLR2、TLR3、TLR4、TLR6、TLR7、TLR8和TLR9在骨髓中均有表达，其中TLR4 mRNA表达水平最高，TLR9 mRNA表达水平最低。本研究建立的检测方法能够成功用于临床样品的检测，为研究马匹TLRs 在mRNA水平的定量分析提供技术平台。

Abstract:

This study aimed to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) method based on SYBR Green Ι fluorescent for detection of horse Toll-like receptor mRNA. According to the gene sequence in conservative region of horse’s Toll-like receptors available in GenBank, the specific primers were designed with β-actin as an internal control to construct Real-time RT-PCR assay. The standard curves showed good linear relationships with the correlation (r²>0.990) and efficiency about 1.0 in the range of 1×10² to 1×10⁸ copies·μL^-1. The melting curve analysis showed that the product was specific to a single peak, and no primer-dimers, with high specificity and sensitivity. The good reproducibility was obtained and the coefficient of variation were less than 3.5% for the intra-assay and inter-assay. The clinical sample test indicated that the TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8 and TLR9 mRNA were transcripted in medulla, and TLR4 mRNA level was highest, but TLR9 mRNA level was lowest. This assay could be successfully used for the detection of clinical samples and provide a technical platform to research horse TLRs at the mRNA levels in the quantitative analysis.

中图分类号:

S821.2

赵一萍，白东义，李蓓，黄金龙，张宇宏，芒来. 马Toll样受体表达水平SYBR GreenⅠ荧光定量RT-PCR检测方法的建立[J]. 畜牧兽医学报, doi: .

ZHAO Yi-ping, BAI Dong-yi, LI Bei, HUANG Jin-long, ZHANG Yu-hong,DUGARJAVIIN Mang-lai. Establishment of a Real-time RT-PCR Assay Based on SYBR Green Ⅰ for Detection of the Expression of Horse Toll-like Receptor Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/

https://www.xmsyxb.com/CN/Y2013/V44/I2/220

参考文献

［1］舒红兵.抗病毒天然免疫［M］.第1版. 北京：科学出版社，2009.

［2］KWON S, VANDENPLAS M L, FIGUEIREDO M D, et al. Differential induction of Toll-like receptor gene expression in equine monocytes activated by Toll-like receptor ligands or TNF-α ［J］. Vet Immunol Immunopathol, 2010, 138:213-217.

［3］FUKATA M, VAMADEVAN A S, ABREU M T. Toll-like receptors(TLRs) and Nod-like receptors (NLRs) in inflammatory disorders ［J］. Semin Immunol, 2009, 21(4):242-253.

［4］UEMATSU S, AKIRA S. Toll-like receptors and innate immunity ［J］. J Mol Med, 2006, 84:712-725.

［5］DUNN-SIEGRIST I, TISSIERES P, DRIFTE G,et al. Toll-like receptor activation of human cells by synthetic triacylated lipid A-like molecules ［J］. J Biol Chem, 2012, 287(20):16121-16131.

［6］SEKI S, NAKASHIMA H, NAKASHIMA M, et al. Antitumor immunity produced by the liver Kupffer cells, NK cells, NKT cells, and CD8⁺ CD122⁺ T cells ［J］.Clin Dev Immunol, 2011, ID868345,doi:10.1155/2011/868345.

［7］MENZIES M, INGHAM A. Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues ［J］.Vet Immunol Immunopathol, 2006,109(1-2):23-30.

［8］IQBAL M, PHIBIN V J, SMITH A L, et al. Expression patterns of chicken Toll-like receptor mRNA in tissues, immune cell subsets and cell lines ［J］.Vet Immunol Immunopathol,2005,104:117-127.

［9］QUINTANA A M, LANDOLT G A, ANNIS K M. Immunological characterization of the equine airway epithelium and of a primary equine airway epithelial cell culture model ［J］.Vet Immunol Immunopathol,2011,140:226-236.

［10］GORNIK K, MOORE P, FIGUEIREDO M, et al. Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva ［J］.Vet Ophthalmol, 2011,14(2):80-85.

［11］KWON S, VANDENPLAS M L, FIGUEIREDO M D, et al. Differential induction of Toll-like receptor gene expression in equine monocytes activated by Tolllike receptor ligands or TNF-α ［J］.Vet Immunol Immunopathol,2010, 138:213-217.

［12］FIGUEIREDO M D, SALTER C E, ANDRIETTI A L P, et al. Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes ［J］.Vet Immunol Immunopathol,2009,131:65-72.

［13］修金生,周伦江,陈如敬,等. 猪流行性腹泻病毒SYBRⅠ实时荧光定量RT-PCR检测方法的建立［J］.中国兽医科学,2012,42(02):160-165.

［14］WONG M L, MEDRANO J F. Real-time PCR for mRNA quantitation［J］.Biotechniques, 2005, 39(1): 75-85.

［15］PFAFFL M W. A new mathematical model for relative quantification in real-time RT-PCR［J］. Nucleic Acids Res, 2001, 29(9): 2002-2007.

［16］韩猛立,黄新,何延华,等.牛Toll 样受体实时荧光定量PCR 检测方法的建立［J］.农业生物技术学报,2011,19,(3):521-529.

［17］LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-^△△Ct method［J］. Methods, 2001,25(4): 402-408.

［18］PEVNER F M, MORAD V, COHEN S M, et al. Toll-like receptors and their ligands control mesenchymal stem cell functions ［J］. Blood, 2007,109:1422-1432.

［19］何晓霞,白海,杨国嵘,等. 人骨髓间充质干细胞中 Toll样受体的表达特征［J］.中国实验血液学杂志, 2009,17(3): 695-699.

［20］张焱如,刘宗正,韦林盖,等. 蒙古马骨髓间充质干细胞的分离培养及多向分化潜能的研究［J］.畜牧兽医学报,2011,42(10):1357-1361.

[1]	任秀娟, 康德措, 苏少锋, 蔺雅楠, 李雅静, 杜明, 白东义, 李蓓, 赵一萍, 芒来. 蒙古马和纯血马免疫相关基因差异表达分析[J]. 畜牧兽医学报, 2023, 54(12): 5020-5032.
[2]	彭宣, 王彤亮, 鲍奕柯, 姚新奎, 孟军, 王建文, 祁居中, 欧阳文, 阿胡·吾拉力别克, 马钶炎, 曾亚琦. 伊犁马体尺与心脏结构和功能参数的关联性分析[J]. 畜牧兽医学报, 2023, 54(11): 4615-4624.
[3]	贾紫洁, 图格琴, 丁文淇, 任秀娟, 刘慧莹, 李欣泽, 翠芳, 芒来, 白东义. 蒙古马全身主要骨骼肌表型谱的构建及比较研究[J]. 畜牧兽医学报, 2023, 54(2): 596-607.
[4]	康周才让, 刘宇, 王敏, 李颖, 毕晓昆, 李媛媛, 凌遥, 赵春江. 利用微卫星分析康西草原马匹种群的遗传多样性和群体结构[J]. 畜牧兽医学报, 2022, 53(5): 1431-1441.
[5]	魏睿元, 赵一萍, 白东义, 韩海格, 王希生, 阿娜尔, 乌英嘎, 芒来, 李旭东. 蒙古马不同耐力运动水平的血浆代谢组特征研究[J]. 畜牧兽医学报, 2022, 53(5): 1442-1454.
[6]	丁文淇, 图格琴, 任秀娟, 陶克涛, 拉希玛, 贾紫洁, 安塔娜, 铁木齐尔·阿尔滕齐米克, 韩海格, 陶娜拉, 芒来, 白东义. 胎儿期与成年期蒙古马骨骼肌肌纤维类型转化研究[J]. 畜牧兽医学报, 2022, 53(1): 88-99.
[7]	张鏻兮, 韩雨薇, 李政, 廖清超, 汤驰, 邓亮. 基于Label-free技术比较马和驴血清蛋白质组分差异[J]. 畜牧兽医学报, 2021, 52(11): 3099-3107.
[8]	潘静, 吴凯峰, 周欢敏, 张焱如. 基于全基因组测序的蒙古马运动相关的正向选择基因筛选[J]. 畜牧兽医学报, 2019, 50(10): 1985-1996.
[9]	任爱珍, 白东义, 赵一萍, 侯娜, 芒来. 基于高速双倍自助法对蒙古马系统发育树的研究[J]. 畜牧兽医学报, 2018, 49(9): 1861-1869.
[10]	白东义, 苏日嘎, 芒来, 乌伊罕, 赵一萍, 李蓓, 格日乐其木格, 张心壮, 陶克涛, 赵若阳, 图挌琴, 青柏, 旭仁其木格. 蒙古马长期高负荷运动训练前后肌肉发育关键基因的表达研究[J]. 畜牧兽医学报, 2018, 49(8): 1617-1624.
[11]	赵珊珊, 肖宁, 李志鹏, 潘庆杰, 李桢, 石德顺, 刘庆友. 马MC1R基因多态位点与毛色相关性分析[J]. 畜牧兽医学报, 2018, 49(8): 1605-1616.
[12]	王建文, 李林玲, 王镜力, 孟军, 曾亚琦, 姚新奎, 辛雅莉. 伊犁马ACTN3基因外显子多态性研究[J]. 畜牧兽医学报, 2018, 49(6): 1299-1306.
[13]	马玉辉, 高利, 李海, 肖建华, 曾亚琦. 伊犁马体尺性状非遗传因素分析及遗传参数估计[J]. 畜牧兽医学报, 2017, 48(10): 1855-1862.
[14]	赵启南, 芒来, 白东义, 赵一萍, 李蓓, 乌尼尔夫, 乌英嘎, 苏日嘎, 敖乳嘎. 蒙古马高负荷运动训练前后转录组差异表达分析[J]. 畜牧兽医学报, 2017, 48(6): 1007-1016.
[15]	潘章源，贺小云，刘秋月，胡文萍，王翔宇，郭晓飞，曹晓涵，狄冉，储明星. 绵羊GDF9基因mRNA、DNA和调控区序列克隆及其在11个品种中遗传多态性检测[J]. 畜牧兽医学报, 2016, 47(8): 1555-1564.