基于G-四联体比色生物传感器检测肉制品中羊源性成分  

A Colorimetric Biosensor Based on G-quadruplex for Detection of Sheep-derived Components

作　　者：王心一 刘榜[1] 谌阳 许文涛[2] 周翔[1] WANG Xinyi;LIU Bang;SHEN Yang;XU Wentao;ZHOU Xiang(Key Laboratory of Agricultural Animal Genetics,Breeding and Reproduction,Ministry of Education,Huazhong Agricultural University,Wuhan 430070,China;College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)

机构地区：[1]华中农业大学,农业动物遗传育种与繁殖教育部重点实验室,武汉430070 [2]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《生物技术进展》2019年第6期641-646,共6页Current Biotechnology

基　　金：国家转基因生物新品种培育重大专项(2018ZX08012-001)

摘　　要：近年来,肉制品掺假现象已成为社会关注的热点,鉴定畜产品中动物源性成分是打击掺假的一种重要技术手段。基于此,利用不对称PCR技术(asymmetric PCR,As-PCR),并结合G-四联体的功能特点,建立了一种G-四联体比色生物传感器以鉴定肉制品中羊源性成分。以羊的基因组DNA为模板,并在限制性正向引物的5′端加上一段G-四联体的反向互补序列,通过As-PCR扩增出大量含有G-四联体序列的单链DNA(single strand,ssDNA),生成的ssDNA在K+的作用下可与氯高铁血红素(hemin)结合形成具有DNA核酶(DNAzyme)活性的G-四联体-hemin复合物。该复合物可催化H2O2与2,2′-联氮-双(3-乙基-苯并噻唑琳-6-磺酸)二胺盐[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt,ABTS 2-]的反应,从而生成绿色的反应产物,结果可用肉眼观察,无需电泳、测序等常规检测步骤。通过对As-PCR反应条件和体系的优化,该生物传感器可实现对肉制品中羊源性成分准确、快速的鉴定,可稳定检测出的混合肉样中羊源性成分的最低质量分数为5%。研究所建立的G-四联体比色生物传感器具有简便、灵敏、可视化、低成本等优点,可用于肉制品中动物源性成分的检测,为肉制品的安全检测提供了强有力的技术支撑。In recent years,the phenomenon of meat adulteration has become a hot topic in society.Identification of animal-derived components in livestock products is one of the important strategies to fight against meat adulteration.Based on this situation,a colorimetric biosensor based on G-quadruplex was established to identify sheep-derived components in meat products by using asymmetric PCR(As-PCR)and combining with the functional characteristics of G-quadruplex.Using sheep genomic DNA as template,and adding a reverse complementary sequence of G-quadruplex to the 5′end of the restrictive forward primer,a large amount of single strand DNA(ssDNA)containing G-quadruplex sequence was amplified by As-PCR,and then the generated ssDNA combined with hemin under the action of K+to form the G-quadruplex-hemin complex with DNAzyme activity.The compound could catalyze the reaction of H2O2 and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt(ABTS 2-)to generate a green reaction product,and the result could be visually observed without conventional detection steps,such as electrophoresis,sequencing,etc.By optimizing the reaction conditions and system of As-PCR,the biosensor could realize accurate and rapid identification of sheep-derived components in meat products,and it could stably detect the minimum mass fraction of sheep-derived components in mixed meat samples to be 5%.The colorimetric biosensor based on G-quadruplex established by the research had the advantages of simplicity,sensitivity,visualization,low cost,etc.And it could be used for detecting animal-derived components in meat products,and it provided strong technical support for the safety detection of meat products.

关 键 词：动物源性成分检测 不对称PCR G-四联体 生物传感器 羊 

分 类 号：TS2[轻工技术与工程—食品科学与工程]