浙江农业学报 ›› 2018, Vol. 30 ›› Issue (11): 1958-1964.DOI: 10.3969/j.issn.1004-1524.2018.11.20

• 生物系统工程 • 上一篇    下一篇

跨膜蛋白39A基因SYBR Green Ⅰ实时荧光定量PCR检测方法的建立及评价

李向茸1, 王兴陇1,2, 李倩1,2, 马瑞仙1,2, 张海霞1, 李琼毅1,2, 马忠仁1, 冯若飞1,*   

  1. 1.西北民族大学 生物医学研究中心,甘肃 兰州 730030;
    2.西北民族大学 生命科学与工程学院,甘肃 兰州 730030
  • 收稿日期:2018-06-07 出版日期:2018-11-25 发布日期:2018-12-21
  • 通讯作者: 冯若飞,E-mail: fengruofei@xbmu.edu.cn
  • 作者简介:李向茸(1987—),女,陕西渭南人,硕士,实验师,主要从事分子病毒学方面研究。E-mail: lxr@xbmu.edu.cn
  • 基金资助:
    国家自然科学基金(31460665);2018年中央高校基本科研业务费资金项目(31920180130);教育部“创新团队发展计划”(IRT_17R88);西北民族大学“双一流”引导专项生物工程特色学科(10018703,1001070204)

Establishment and evaluation of SYBR Green Ⅰ real-time PCR for quantitatively detecting TMEM39A gene

LI Xiangrong1, WANG Xinglong1,2, LI Qian1,2, MA Ruixian1,2, ZHANG Haixia1, LI Qiongyi1,2, MA Zhongren1, FENG Ruofei1,*   

  1. 1. Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China;
    2. Life Science and Engineering College, Northwest Minzu University, Lanzhou 730030, China
  • Received:2018-06-07 Online:2018-11-25 Published:2018-12-21

摘要: 为建立一种快速检测跨膜蛋白39A(TMEM39A)基因的方法,根据GenBank中登录的不同种属TMEM39A基因序列的保守区域,设计并合成1对荧光定量通用性引物,经过条件优化,建立了检测TMEM39A基因的SYBR Green Ⅰ实时荧光定量PCR方法。结果显示:标准品模板在3.937×108~3.937×103拷贝·μL-1范围内呈良好的线性关系,R2可达0.999;该方法特异性较好,检测灵敏度可达3.937×102 拷贝·μL-1;组内和组间变异系数均小于1%,具有较高的重复性和稳定性;利用该方法能够检测出不同细胞中的TMEM39A含量,具有种属通用性。该研究方法的建立为临床上提供了一种TMEM39A的快速检测和定量分析技术。

关键词: 跨膜蛋白39A, SYBR Green I实时荧光定量PCR, 特异性, 灵敏性

Abstract: In order to establish a rapid simple and reliable method for TMEM39A gene detection, a SYBR Green Ⅰ real-time PCR method was developed with a pair of primers designed according to the published gene sequence of TMEM39A in GenBank, and the reaction conditions were optimized. The results showed that the Ct value of the assay linearly related to the standard plasmid in the range of 3.937×108-3.937×103 copies·μL-1 and the standard curve correlation coefficient R2 was 0.999. It also had good specificity and sensitivity. The reproducibility was high, and the variation coefficients of intra-assay and inter-assay were less than 1%. This method could detect the content of TMEM39A gene in different cells, so its generality was good. In this study, a rapid and accurate method for quantification of TMEM39A gene by SYBR Green Ⅰ real-time PCR was successfully developed.

Key words: transmembrane protein 39A (TMEM39A), SYBR Green I real-time quantitative PCR, specificity, sensitivity

中图分类号: