实时荧光LAMP法检测转基因猪的标记基因hCD39

doi:10.11843/j.issn.0366-6964.2017.03.018

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (3): 538-543.doi: 10.11843/j.issn.0366-6964.2017.03.018

实时荧光LAMP法检测转基因猪的标记基因hCD39

李津¹，刘巍¹，李俊超²，金红岩¹，李刚^1*

(1.中国农业科学院北京畜牧兽医研究所农业部兽用药物与兽医生物技术科学观测实验站，北京100193; 2. 中国人民解放军总医院心血管内科，北京 100853)

收稿日期:2016-08-30 出版日期:2017-03-23 发布日期:2017-03-23
通讯作者: 李刚，教授，E-mail: ligang03@caas.cn
作者简介:李津(1988-)，男，河北冀州人，硕士生，主要从事生物制药与药物分析方面的研究，E-mail:lijin186@eyou.com,Tel：010-59613515
基金资助:
国家转基因重大专项（2014ZX0801203B-002）；国家自然科学基金（31472203）；国家科技支撑计划(2013BAD12B05)；FAO/IAEA 国际合作项目（17453/D32025）

Real-time Fluorescence Loop-mediated Isothermal Amplification for Detection of hCD39 in Transgenic Pig

LI Jin¹，LIU Wei¹，LI Jun-chao²，JIN Hong-yan¹，LI Gang^1*

(1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Department of Cardiovascular, China PLA General Hospital, Beijing 100853, China)

Received:2016-08-30 Online:2017-03-23 Published:2017-03-23

摘要/Abstract

摘要：

利用ESE-Quant tube scanner监测平台，建立了一种基于实时荧光LAMP技术的hCD39基因快速检测方法。针对hCD39基因设计6条特异性引物，在63 ℃条件下，进行核酸扩增反应45 min。在扩增前加入SYBR Green Ⅰ染料作为反应的指示剂，以SYBR Green Ⅰ染料的荧光强度作为结果判定标准。该方法灵敏度高，最小可检测到3 fg•μL^－1；特异性好，与转基因猪中的标记基因hCD46、LEA29Y均无交叉反应；并且可对口腔拭子标本进行检测。因此，研究建立的实时荧光LAMP法，操作简单，反应快速，灵敏度高，特异性好，并且可以对猪活体进行无损伤采样检测，适合在现场快速简便的操作。

Abstract:

Using ESE-Quant tube scanner testing platform, we established a real-time fluorescence loop-mediated isothermal amplification (RTF-LAMP) method for rapid detection of hCD39. The method employed a set of six specially designed primers for amplification of nucleic acid under isothermal conditions at 63 ℃ for 45 min. The amplification process of LAMP was monitored by the addition of SYBR Green Ⅰ dye prior to amplification. The fluorescence intensity of SYBR Green Ⅰ dye was used as the standard of the results. Results of the study showed that the sensitivity of the method was 3 fg•μL^－1, and there was no cross-reactivity with the genes of related and unrelated detected, such as hCD46, LEA29Y. More importantly, this method could be completed testing of buccal swabs specimens of transgenic pigs. The purpose of our study was to develop a simple, economical, rapid method with good sensitivity and specificity. The method could be no damage to the living body samples for testing, suitable for quick and easy on-site operation.

中图分类号:

S858.91

李津，刘巍，李俊超，金红岩，李刚. 实时荧光LAMP法检测转基因猪的标记基因hCD39[J]. 畜牧兽医学报, 2017, 48(3): 538-543.

LI Jin，LIU Wei，LI Jun-chao，JIN Hong-yan，LI Gang. Real-time Fluorescence Loop-mediated Isothermal Amplification for Detection of hCD39 in Transgenic Pig[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 538-543.

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/10.11843/j.issn.0366-6964.2017.03.018

https://www.xmsyxb.com/CN/Y2017/V48/I3/538

参考文献

［1］NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA［J］. Nucleic Acids Res, 2000, 28(12): e63.
［2］SEYRIG G, STEDTFELD R D, TOURLOUSSE D M, et al. Selection of fluorescent DNA dyes for real-time LAMP with portable and simple optics［J］. J Microbiol Methods, 2015, 119: 223-227.
［3］张丽娜，胡成进. 环介导等温扩增技术在病原微生物快速诊断中的应用［J］. 生物技术通讯, 2015, 26(6): 886-890.
ZHANG L N, HU C J. Application of Loop-mediated isothermal amplification technique for rapid diagnosis of pathogenic microorganisms ［J］. Letters in Biotechnology, 2015, 26(6):886-890. (in Chinese)
［4］匡燕云，李思光，罗玉萍. 环介导等温扩增核酸技术及其应用［J］. 微生物学通报, 2007, 34(3): 557-560.
KUANG Y Y, LI S G, LUO Y P. Loop-mediated isothermal amplification method for detection of nucleic acids and its application［J］. Microbiology, 2007, 34(3): 557-560. (in Chinese)
［5］ZHU H T, YU L, LYU Y , et al. Optimal pig donor selection in islet xenotransplantation：current status and future perspectives［J］. J Zhejiang Univ Sci B, 2014, 15(8): 681-691.
［6］潘登科，冯冲，龙川，等. 用于糖尿病治疗的多基因修饰小型猪［C］. 2013中国器官移植大会, 杭州, 2013.
PAN D K, FENG C, LONG C, et al. Multi gene modified minipig for diabetes treatment ［C］. 2013 Chinese Transplant Congress, Hangzhou, 2013. (in Chinese)
［7］李伟，李刚，范晓娟，等. 快速检测小反刍兽疫病毒RT-LAMP方法的建立［J］. 中国预防兽医学报, 2009, 31(5): 374-378.
LI W, LI G, FAN X J, et al. Establishment of a rapid method for detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification ［J］. Chinese Journal of Preventive Veterinary Medicine, 2009, 31(5): 374-378.（in Chinese）
［8］COOPER D K, DORLING A, PIERSON R N 3rd, et al. Alpha1,3-galactosyltransferase gene-knockout pigs for xenotransplantation: Where do we go from here?［J］. Transplantation, 2007, 84(1): 1-7.
［9］TAKENAKA M C, ROBSON S, QUINTANA F J. Regulation of the T cell response by CD39［J］. Trends Immunol, 2016, 37(7): 427-439.
［10］SCALEA J, HANECAMP I, ROBSON S C, et al. T-cell-mediated immunological barriers to xenotransplantation［J］. Xenotransplantation, 2012, 19(1): 23-30.
［11］GODEHARDT A W, SCHILLING-LEISS D, SANZENBACHER R, et al. Xenogeneic cell therapeutics: Treatment of type 1 diabetes using porcine pancreatic islets and islet cells［J］. Bundesgesundheitsblatt, Gesundheitsforschung, Gesundheitsschutz, 2015, 58(11-12): 1281-1288.(in German)

实时荧光LAMP法检测转基因猪的标记基因hCD39

Real-time Fluorescence Loop-mediated Isothermal Amplification for Detection of hCD39 in Transgenic Pig

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics

本文评价