基于RT-PCR动物源性食品掺假量化判定技术及其相关理论研究

The incidents of meat species adulteration in meat products, such as the "beef extract in Anhui Province", frequently happened in recent years. The outdated detection technologies and standards have caused large hidden safty troubles in our country. Especialy for animal-derived food, there is no mature detection method currently. The difficulty of analytical methods of detecting meat species in animal-derived food lies to how to make a distinction between "sample contamination" and "malicious adulteration". The "species-specific combined with universal primers/probes" RT-PCR method which was used to determine the percentage content of animal-derived components developed by our research group is expected to exceed the difficulty and some research foundation has been made. Relevant technologies and its basic theoretical researches will be carried out around a series of key issues, such as properties control of prmers/probes, competitive reaction kinetics of duplex RT-PCR, the principles of DNA template heated fracture and confidence interval setting and so on by RT-PCR, cDNA array, thermal processing, perl programming, mathematical modeling technologies. The mature RT-PCR-based species-specific DNA and tissue-specific mRNA quantitative detection technology and associated primer/probe design technology system for adulteration identification will be established in order to provide technical supports and theoretical references for the regulatory authorities.

近年来，包括"安徽牛肉膏"肉制品掺假事件在内的食品质量与安全事件频发，而我国相应的检测技术、标准较为落后，造成了较大安全隐患。特别是针对肉类等动物源性食品掺假行为尚无成熟的鉴别技术可用，难点在于无法区别"样品污染"与"恶意添加"行为，更无法区分杂碎成分。本团队提出的"物种特异性、通用性引物/探针联用测定样品中动物源性成分百分比含量的RT-PCR技术"有望突破该难点，目前已取得一定工作基础。拟围绕遇到的诸如引物/探针特性调控、双重RT-PCR竞争性反应动力学、DNA模板受热断裂规律、置信区间设置等关键问题，应用RT-PCR、cDNA微阵列、热加工、perl语言编程、数学建模等多学科交叉技术开展相关技术及其基础理论研究，建立起成熟的适用于掺假鉴别，基于RT-PCR的物种特异性DNA和组织特异mRNA定量检测技术及其配套的引物/探针设计技术体系，为监管部门提供技术支撑和相关技术研发提供理论参考。

近年来，肉制品掺假事件频发，而国内缺乏能够准确甄别掺假行为严重程度的技术，为监管部门执法造成巨大困难。本研究应用RT-PCR、perl语言编程、数学建模等多学科交叉技术，建立了基于RT-PCR的动物源性成分定量检测及配套的引物/探针设计技术，具体研究内容和结果包括：（1）研究了碱基错配、探针位置对RT-PCR的影响规律及DNA模板的受热断裂规律。构建了错配反应“ΔCt值-非特异性扩增”预测模型，发现异型错配较易发生非特异性扩增，同型错配则较难发生；探针与引物序列重合过多或距离过远时，难以发生RT-PCR反应；加热方式对线粒体DNA断裂影响较大，加压加热高于水浴加热，DNA片段长度越长受热断裂的概率也越大。（2）建立了以物种特异性引物和通用引物为基础的猪、牛、羊、鸡、鸭、鹿、驴源性成分百分比含量测定的RT-PCR方法，包括适用于单重和双重PCR的引物、探针体系，反应试剂配比、反应条件和结果计算方法。该方法体系具有良好的特异性、准确度。（3）通过研究肉制品干肉重与RT-PCR反应Ct值的一一对应关系，建立了基于“干肉重-Ct值”标准曲线的猪、牛、羊、鸡、鸭、驴、鹿绝对定量方法。（4）对“循环数-荧光信号”数据进行拟合得到Gompertz模型方程，根据Ct值定义、“循环数-荧光信号”数据及模型方程可计算出Ct值，实现了Ct值计算的标准化，有效排除了仪器之间带来的结果差异和自带软件的诸多弊端。（5）建立了一种基于5重RT-PCR熔解曲线分析的肉制品掺假鉴别方法，可同时检测出猪、牛、绵羊、山羊、鸡、鸭、狗、狐、貉9种源性成分。方法具有良好的特异性及灵敏度，单物种DNA检出限为0.001~1 ng，多物种混合DNA检出限为0.1 ng，与定量检测技术配合使用可解决动物源性成分掺假判定难的现状。本项目建立的动物源性成分定量检测技术，将为肉制品掺假定性、量刑难题提供可靠的技术手段和执法依据，对保护消费者和肉制品企业利益具有重要意义。