Abstract: 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 (ABTS2-) 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.

Key words: detection of animal-derived components, asymmetric PCR, G-quadruplex, biosensor, sheep

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\[2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt，ABTS2-\]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。

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