羊肉及其制品中掺假动物源性成分数字PCR技术精准定量研究

发布时间：2018-01-11 03:19

本文关键词：羊肉及其制品中掺假动物源性成分数字PCR技术精准定量研究　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

更多相关文章： 数字PCR 羊肉 精准定量 真伪鉴别 掺假

【摘要】：近年来,食品欺诈问题日益突出,肉制品掺假现象尤为严重。前几年欧洲爆发的"马肉风波"揭露了多国肉制品的掺假现象;我国同样存在不法分子将廉价肉掺入高价肉中,以获取巨额利润的经济利益驱动型掺假现象。在肉类产业中,肉羊是我国畜牧业的重要支柱产业之一,消费需求旺盛。然而,用鸡、鸭和猪肉掺入甚至替代羊肉的事件屡见不鲜。这些问题的出现严重干扰了我国畜肉产业,制约了肉品质量的提升,并严重损害了消费者的权益,甚至涉及宗教信仰等问题。因此,提升羊肉的真伪鉴别能力对于保障居民的食肉安全有着重要的现实意义。目前,羊肉的真伪鉴别检测大多停留在定性和半定量阶段,目前尚无精准的定量检测方法,因此无法确定掺假成分是有意添加还是无意污染,更无法作为食品掺假犯罪中量刑依据的定量检测标准方法推广。因此,亟待建立一种精准、快速、高通量的定量方法,对羊肉及其加工品中不同动物源性掺假成分进行精准定量检测。本论文基于微滴式数字PCR技术,建立了针对羊肉及其多种动物源性掺假成分的定性及精准定量检测方法,具体研究工作如下:1.针对羊肉及其掺假物的定性检测,选取单拷贝核基因作为检测靶标,通过筛选和优化各物种的特异性引物、探针和数字PCR体系,建立了基于数字PCR技术的羊肉及其掺假物定性检测方法;对方法特异性、稳定性以及灵敏度的验证结果表明该检测体系可以对掺假物种含量为0.1%的羊肉样品实现稳定检测,且灵敏度高、特异性好。2.针对纯肉制品的精准定量,通过理论推导获得单位质量两种肉基因拷贝数之比的倍增系数,并进行实验验证,据此将羊肉和其他掺假肉的基因拷贝数转化为质量分数,最低定量检测限达到1%(w/w),模拟不同条件对肉制品进行处理,结果表明定量方法的准确度和重复性不受样品部位、超高压、加热和冷冻处理等条件的影响。3.针对含有食品配料和添加剂羊肉制品的精准定量,通过在样品中加入内标(火鸡)肉种,制备标准样品,绘制标准曲线,计算得到各物种基因拷贝数与质量的转化公式,进而计算出肉的质量,最低定量检测限为1%(w/w),模拟不同条件对肉制品进行处理,结果表明定量方法的准确度和重复性不受添加剂和配料的种类、样品部位、超高压、加热和冷冻处理条件的影响。4.针对羊肉中同时掺杂多种动物源性成分情况下的定量检测,在单一物种定量检测方法的基础上,建立基于倍增系数法的多重数字PCR定量检测体系,包括羊肉中同时掺杂鸡、鸭和猪肉的四重数字PCR定量检测体系和羊肉中同时掺杂狐狸、水貂和貉肉的四重数字PCR同时定量检测体系;通过在混合样品中加入内标(火鸡),计算掺假物种与内标肉种的比值,建立了基于内标法的五重数字PCR定量检测体系。综上所述,本论文基于微滴式数字PCR技术建立的两种肉制品中物种成分含量分析方法,可分别实现对羊肉中多种掺假物种的定性筛查、单一掺假成分的精准定量检测以及多种动物源性成分同时精准定量检测,从而初步建立了羊肉质量检测的表征属性识别技术体系,该体系不仅可以定性和定量羊肉中掺杂的动物源性成分,也可以应用于其他肉类及其他食品掺假的定性和定量检测,为羊肉及其制品的市场监管和相关执法提供有力的技术保障。
[Abstract]:In recent years, food fraud problems have become increasingly prominent, meat adulteration phenomenon is particularly serious. A few years ago the European outbreak of "horsemeat" reveals the phenomenon of adulterated multinational meat products; China also has the criminals will be cheap meat meat mixed with high price, in order to obtain huge profits driven by economic interests in the adulteration. The meat industry, meat is China's animal husbandry is one of the important pillar industry, consumer demand. However, with chicken, duck and pork mixed with mutton or even replace the event. It is often seen. these problems serious interference in the meat industry, restricting the meat quality improvement, and serious damage to the interests of consumers and even religion and other issues. Therefore, enhance the ability to identify the authenticity of mutton has important practical significance for the protection of residents food safety. At present, the authenticity of mutton are qualitative detection Semi quantitative and quantitative detection method, there is no accurate current, therefore unable to determine the adulteration is intentionally added or not pollution, not as the extension of the standard method for quantitative detection of adulteration of food crime sentencing basis. Therefore, urgent need to establish an accurate, rapid, high-throughput quantitative method, accurate quantitative detection of different animal the source of mutton and its processed products of adulteration. The droplet digital PCR technology based on the established detection method for qualitative and a variety of animal derived mutton adulteration and accurate quantitative, specific research work is as follows: 1. for mutton and adulterated qualitative detection, selection of single copy nuclear gene as target detection. Through the screening and optimization of specific primers for each species of the probe and the digital PCR system was developed to detect mutton and its adulterant qualitative digital PCR technology based on; The specificity of the method, stability and sensitivity of the verification results show that the detection system of adulteration species content of 0.1% mutton samples to achieve stable detection, high sensitivity, good specificity and accurate quantitative.2. for pure meat products, obtain copies of unit quality of two kinds of meat gene of the ratio of the number of multiplication coefficient by theoretical derivation, and based on the experiment, mutton and other meat adulteration gene copy number into the mass fraction, the minimum quantitative detection limit reached 1% (w/w), the simulation of different conditions on meat processing, results showed that the accuracy and repeatability of quantitative method from the sample site, ultra high pressure, heating and freezing conditions effect of.3. for accurate quantification of containing food ingredients and additives of mutton products, in the sample by adding internal standard (Turkey) meat, the preparation of standard sample, drawing the standard curve, the calculated A gene copy number and mass conversion formula, and then calculate the meat quality, the lowest limit of quantification was 1% (w/w), the simulation of different conditions on meat processing, the result show that the type, accuracy and repeatability of quantitative methods without additives and ingredients sample parts, high pressure, heating and freezing treatment the effect of.4. on various conditions of quantitative animal derived ingredients and mutton in the doping test, based on the quantitative detection method of single species, the establishment of multiple digital PCR quantitative detection system based on the multiplier method, including sheep meat and chicken and the fox four doped, doped heavy digital PCR quantitative detection system and mutton duck and pork, four mink and racoon dog meat and digital PCR quantitative detection system; through the mixed samples with internal standard (Turkey), calculation of species and the internal standard of meat adulteration ratio value is established based on Five digital PCR quantitative detection system of internal standard method. To sum up, this paper analysis of species composition content of two kinds of meat products to establish the droplet digital PCR technology based on can be realized respectively for qualitative screening multiple species adulteration in Mutton adulteration, single precision quantitative detection and a variety of animal derived ingredients at the same time accurate quantitative detection, we preliminarily established the system characterization of attribute recognition technology of mutton quality detection, the system can not only components of animal origin doping of lamb in the qualitative and quantitative, qualitative and quantitative detection can also be applied to other meat and other food adulteration, to provide strong technical support for mutton and its products and related market supervision law enforcement.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS251.7

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