液相色谱串联质谱对掺假牛肉的鉴别及定量研究

本文以差异蛋白质组学为理论研究基础,利用高效液相色谱串联质谱(HPLC-MS/MS)实现了对猪肉或鸡肉掺假的牛肉的定性鉴别及定量分析。首先采用高分辨质谱仪(nLC-QE)分别找出牛肉、猪肉和鸡肉的相对专属性多肽链,并对其进行二级扫描,得到了三个物种的特异性离子对,进一步利用HPLC-MS/MS的多反应监测模式(MRM)对掺杂猪肉或鸡肉的牛肉进行特异性离子对验证,分别选择了牛肉、猪肉和鸡肉的9、8和7条多肽进行定性研究;并与聚合酶链式反应(PCR)技术进行比对,得到的结果一致;此外将鸡肉和猪肉以0.5、1.0、5.0、10.0、25.0%的比例掺杂于牛肉中,各选择3组离子对进行定量分析,其各自的线性相关系数均大于0.99,最低定量限为0.5%。由此表明,液相色谱串联质谱的方法可以快速地对牛肉中的猪肉和鸡肉进行鉴别并能准确定量分析。     

基于欧式距离法或因子化法的近红外光谱技术对牛肉掺假鉴定的研究

利用近红外光谱技术结合欧氏距离法或因子化法建立牛肉中掺入猪肉和鸡肉的快速、准确的技术方法,从而为保证牛肉制品品质和安全提供强有力的检测手段;以牛肉和掺假肉(掺入猪肉和鸡肉)按照相同程度进行均匀搅碎,按照掺假肉10%为梯度增量进行制备掺假肉,共分为10类样本(10%至100%),采用单通道,光谱范围为12000cm^-1~4000cm^-1,扫描次数为64次,特征波段为7351.92cm^-1~6824.83cm^-1、5578.24cm^-1~5075.21cm^-1,预处理方式为一阶导数或二阶导数+矢量归一化+9点平滑,结果表明:猪肉和鸡肉掺假因子化法的判别率分别为94%和96.67%;因子化法可以试现对牛肉掺假进行定性分析鉴定。     

应用微滴式数字聚合酶链式反应定量检测牛肉制品中的猪源性成分

参照GB/T 25165—2010《明胶中牛、羊、猪源性成分的定性检测方法 实时荧光聚合酶链式反应法》,以牛的生长激素（growth hormone,GH）基因和猪的朊蛋白（prion protein,PRNP）基因2 种核基因为靶基因合成特异性引物和探针。理论推导单位质量牛肉的GH基因与猪肉PRNP基因拷贝数之比为一固定值,通过微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction,ddPCR）方法对该固定值进行检测和验证,并以该固定值对牛肉/猪肉人工混合样本和市售牛肉制品中的猪肉成分进行定量检测。结果表明：该方法具有良好的准确性及可重复性;牛肉中掺杂猪肉质量分数在5%～99%范围内时,检测结果绝对误差小于1.28%,变异系数小于6.5%,猪肉成分的回收率为99.09%～102.80%;20 份牛肉制品中,4 份检出猪肉成分,其中3 份的相对含量为29.19%～98.15%,1 份为0.12%（低于本方法检出限5%）。因此,ddPCR方法可以较好地应用于牛肉制品中掺杂猪肉成分的定量检测。     

用Cytb基因序列分析鉴定肉制品掺假

目的采用Cytb基因进行肉制品真伪鉴定的尝试性探索。方法随机采集21份肉制品样品,首先采用CTAB法方法提取样品DNA,基于Cytb基因的通用引物对21份样品DNA进行PCR扩增,把扩增产物进行测序,然后采用DNAMAN软件进行序列的比对分析。结果 CTAB方法适合肉制品的DNA提取。10份牛肉样品中9份与标注吻合,1份检测出是猪肉;2份羊肉样品中1份与标注吻合,1份检测是猪肉;2份猪肉样品中1份与标注吻合,1份检测是鸡肉;1份鸡肉样品与标注吻合;6份牦牛肉样品中2份与标注吻合,4份检测是水牛肉。结论用Cytb基因序列分析比对方法适合肉制品的真伪鉴定,市场上肉制品存在掺假问题。     

荧光定量PCR方法检测肉制品中鳄鱼源性成分

针对鳄鱼色素细胞b基因序列设计特异性引物,建立一种快速、特异、灵敏的鳄鱼源性成分检测方法。以常见羊肉、牛肉、猪肉、鸡肉、鸭肉、鱼肉、虾肉为参考动物物种作特异性检测,并经检出限和灵敏度实验验证其可行性。结果表明：该方法能够有效对鳄鱼源性成分进行快速检测,具有较强的特异性,鳄鱼组分DNA的检出限可达0.001 ng/μL,灵敏度可达0.01 %。通过加工肉制品的检测验证,该方法拥有特异性强、灵敏度高的特点,可以用于加工肉制品中鳄鱼源性成分的检测。     

广东省牛羊肉及其制品中掺杂掺假情况的调查分析

目的对广东省内牛、羊肉及其制品中掺杂掺假情况进行风险监测,从而为监管部门的后续监督管理提供指导性意见。方法使用特异性引物和探针,对广东省市场上出售的牛、羊肉及其制品进行动物源性成分鉴定。并与标签明示肉源进行比对,确认掺假类别。结果共检验50份样品,其中牛肉25份,羊肉8份,混合肉类17份。检出10份掺假肉食品,总掺假率为20.0%。掺假样品均为牛肉制品,羊肉制品为未发现掺假情况。结论用猪肉和鸡肉进行肉类的掺假是目前主要的掺假手段。混合肉类制品在标签明示肉类成分方面比较混乱,部分样品检出标签未标示的肉类成分。进一步开展肉制品的掺假检测具有重要的社会意义。     

2013年苏州地区肉及其制品掺假情况调查

了解苏州地区肉及其制品的掺假情况,通过对肉类种源与标签明示肉源进行比对,鉴别摻假食品,为加强食品标签管理提供依据。方法 运用自建的动物源性食品种源判定Taqman实时荧光PCR检测体系对苏州地区的肉及其制品进行种源判定,与标签明示肉源进行比对,鉴别摻假食品。结果 本次调查共检验涉及32个生产单位的90份样品,总不符合率为25.6%(23/90)。检测的44份牛肉及其制品中有12份与标签不符,8份用猪肉部分替代牛肉,1份以鸭肉部分代替牛肉进行销售;此外有3份不含有牛肉成分,存在猪、鸡、鸭源性肉类之外的肉类成分。共检测羊肉及其制品16份,有2份用鸭肉代替羊肉出售,3份羊肉样品中掺入了部分猪成分,其中1份样品还存在单个样品掺杂两种外源肉类的现象(猪源性和鸭源性)。检测猪肉及其制品19份,其中2份样品含有标签未注明的鸡肉成分。在所检测的11份混合肉类样品中有4份成分与标签不符,主要是以廉价的鸡肉取代/部分取代相对高价的牛肉和猪肉。结论 肉制品掺假情况明显,用猪肉、鸭肉部分代替牛肉和羊肉仍是主要的掺假手段,牛肉掺假样品主要是熟制牛肉制品,而火锅食用羊肉卷样品则是羊肉掺假高危品,开展肉制品摻假检测对规范肉制品市场具有积极意义。此外,3份未知种源成分的牛肉样品提示在现有检测基础上还需扩大检测范围,防患于未然。     

利用多重PCR方法检测牛肉中的掺假肉

应用多重PCR方法鉴别牛肉中掺杂其他肉类。通过查阅相关文献,确定牛肉、猪肉、鸡肉、鼠肉的特异性片段设计引物。牛肉样品通过基因提取试剂盒提取组织DNA,然后加入相应特异性牛肉、猪肉、鸡肉、鼠肉引物和反应试剂,采用多重PCR法同时检测牛肉中掺杂鸡肉、猪肉、鼠肉任意三种肉类混合样品。试验建立的复式PCR方法特异性强,操作简便,对于牛肉中掺杂鸡肉、猪肉、鼠肉的检出限均达到1%(W/W)掺杂量。     

基于线粒体16SrRNA基因鉴别畜禽肉中5种动物源性成分

目的:建立一种快速、特异、灵敏的动物源性成分检测方法。方法:以线粒体16S r RNA基因序列为靶位点设计特异性引物,以常见畜禽包括羊、牛、猪、兔、鸡、鸭、鹅、鸽、鹌鹑等参考动物为研究对象,提取肌肉组织DNA,进行PCR扩增,设计筛选羊肉、牛肉、猪肉和兔肉特异性引物对,并进行引物特异性研究。结果:选择的特异性引物能够有效地对包括羊肉、牛肉、猪肉、兔肉以及鸡肉在内的5种动物源性成分进行快速检测,方便简洁。结论:该方法可以快速、准确地检测畜禽肉中羊肉、牛肉、猪肉、兔肉和鸡肉成分。     

一种基于双重ddPCR的肉制品中牛源性成分的定量分析方法

将低价肉类原料掺入高价肉制品是一种典型的肉类掺假方式，为准确鉴别牛肉及牛肉制品的真伪，建立一种基于双重微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）的牛源性成分定量分析方法。通过对14 种动物的Beta-actin单拷贝基因序列进行分析，设计牛源性特异性引物、探针与动物源性成分通用引物、探针并建立双重ddPCR体系，推导出牛源性成分质量与其特异性扩增拷贝数之间的计算公式，对牛源性成分进行定量检测该方法得到牛源性成分质量M牛（mg）与其特异性扩增拷贝数浓度C牛（copies/μL）之间的计算公式为M牛=0.033C牛＋2.37，对已知牛肉质量的混合肉样品与不同部位的牛肉样品进行检测，结果显示牛肉定量检测值与实际质量基本一致。同时拷贝数相对含量可辅助判断肉制品中是否存在非牛源性的其他动物源性成分掺假。对市售样品的检测发现，存在目标肉样含量不达标以及不同程度的动物源性成分等掺假现象，说明该检测方法可为牛肉制品掺假量化判定和混合源性产品分级鉴定提供技术支撑。     

Identification of chicken,duck, pigeon and pig meat by species-specific markers of mitochondrial origin

In the present study, PCR based method for meat species identification of chicken, duck, pigeon and pig was achieved by developing species-specific markers. Using mitochondrial sequences species-specific primers were designed and the sizes of them were 256 bp, 292 bp, 401 bp and 835 bp for chicken, duck, pigeon and pig, respectively. The species-specific PCR products were sequenced to confirm the specificity of the product amplified. These markers were subsequently tested for cross amplification by checking them with beef, mutton, chevon, pork, rabbit, chicken, duck, turkey and pigeon meat. DNA markers developed in this study can help identify the species of fresh, cooked and autoclaved meat of chicken, duck and pigeon and fresh and cooked meat of pig. The process of identification is simple, economical and quick as compared to other methods such as RAPD, PCR-RFLP and sequencing method of species identification.     

基于COI序列的DNA微条形码技术鉴别熟肉制品中11种肉掺假的研究

建立并优化了基于COI序列的DNA微条形码技术（mini-barcoding）检测熟肉制品中11种常见肉类掺假的方法。样品经超声与真空冷冻干燥处理,提取DNA模板和PCR扩增后,目标扩增物经切胶纯化后进行克隆测序,并将测序结果提交GenBank数据库Blast比对。筛选出适合猪、牛、羊、鸡、鸭、鸽子、马、驴、鹅、兔、鼠11种肉类的扩增的通用引物COI-A,对PCR扩增条件进行优化,并建立18个掺假模式,对低经济价值肉类（猪、鸡、鸭、马、驴、鼠）的掺入的最低比例进行考察。结果表明:11种熟肉的DNA经通用引物COI-A扩增后,其扩增效率均为100%,18个掺假模式中,牛肉和羊肉中掺入6种低经济价值肉类的最低检出比例为5%,而掺入鸡肉的最低检出比例为10%,而鹅肉、鸽子肉和兔肉的掺假模式中最低检出比例为10%;利用本方法检测30批次市售样品,发现有18批次的熟肉制品存在掺假情况。该方法前处理简单,灵敏度合适,重现性好,可作为高经济价值熟肉制品中掺假低经济价值肉类的有效检测方法。     

非标记蛋白定量方法在掺假肉鉴别中的应用

目的建立一种非标记蛋白定量法鉴别牛肉或羊肉中是否掺假。方法采用四极杆串联飞行时间高分辨质谱分析各种肉类蛋白酶解之后的样品,找到每种肉类特异的肽段,再用串联四极杆质谱测定特异肽段的定量结果,判定样本中是否有这种肉类存在。结果在测定的牛肉、羊肉、猪肉、鸡肉和鸭肉样本中,均找到每种肉类的特异肽段5～7条,从每种肉中选择3条肽段进行定量分析,可以很好地鉴别出牛羊肉中掺杂的其他肉类。结论该方法操作简便,结果可靠,灵敏度高,可用于在牛肉或羊肉中掺杂其他肉类的鉴别。     

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods

The method performance characteristics of commercially available PCR kits for animal species identification were established. Comminuted meat products containing different levels of pork were prepared from authentic beef, chicken, and turkey. These meat products were analysed in the raw state and after cooking for 20 min at 200 °C. For both raw and cooked meats, the PCR kit could correctly identify the animal species and could reliably detect the addition of pork at a level below 0.1%. A survey of 42 Turkish processed meat products such as soudjouk, salami, sausage, meatball, cured spiced beef and doner kebap was conducted. Thirty-six samples were negative for the presence of pork (< 0.1%) and four were found to be correctly labelled as containing pork. However, one sausage sample was labelled as containing 5% beef, but beef DNA was not detected and a meatball sample labelled as 100% beef was found to contain chicken. Another turkey meatball sample was predominantly chicken.     

基于实时荧光定量PCR和数字PCR的肉制品中牛源性成分检测

为了能够快速有效的检测出肉制品中牛源性成分,采用实时荧光定量PCR和数字PCR检测方法,检测肉制品中牛源成分。先依据牛肉单复制基因组特异区域,通过多序列比对设计特异性引物与探针,再利用苯酚-氯仿法提取标准品基因组DNA,确立实时荧光定量PCR和数字PCR两种方法的反应条件,测定DNA模版纯度及浓度可得实验提取的DNA溶液不含杂质,在此基础上构建牛源性成分荧光定量PCR标准曲线,检测两种方法的特异性、抗干扰性与肉制品中牛源性成分。分析实验结果可知,引物与探针在两种方法中均对牛肉有荧光信号显示,两种方法具有牛源性特异性,检测数值与实际样品数值基本一致,且两种方法抗干扰性较好,当存在其他肉类干扰时,仍可准确监测出牛源性,两种方法的最大误差分别为3.8%和4.0%;可定量检测不同肉制品中牛源性成分,两种方法均未检测出样品10牛肉丸中牛源性成分,验证了市面上确实存在假肉情况。说明所用方法能够准确、快速地检测出肉制品中的牛源性成分,适用于市场中肉制品的检测,对保障消费者的权益和健康具有十分重要的意义。     

基于多元统计分析的牛排掺假定量判别及差异分析

结合市场对食品中不同成分的定量分析的需求，模拟不同比例混合样品，利用高分辨质谱进行数据采集及分析，分别考察主成分分析、偏最小二乘判别分析和正交偏最小二乘判别分析（orthogonal partial least squares-discrimination analysis，OPLS-DA）统计方法对不同混合比例的样品数据进行分析。相比之下，OPLS-DA模型具有更好的判别效果。通过进行S-Plot分析，分别筛选出25?条猪源多肽及牛源多肽，经线性拟合分析，其中牛源的10?条多肽和猪源的6?条多肽的线性相关系数R2大于0.99，并开展了真实性样品的验证实验，证明筛选的多肽可用于预计样品中的成分含量。这项研究建立了一种量化牛排的方法，并为筛选差异显著性的定量多肽提供新思路。     

Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.     

Comparison of flavor changes in cooked-refrigerated beef, pork and chicken meat patties

Beef and pork longissimus dorsi (LD) and semimembranosus (SM) and chicken breast (B) and thigh (T) muscles excised 24 h postmortem were ground by muscle/species group, formed into patties, pan-fried, refrigerated for 0, 3 or 6 days, and evaluated by a trained sensory panel for intensity of specific flavors. The rate of decline in species-specific natural meat flavor intensity and the rate of increase in "cardboard" (CBD) flavor intensity during the first half of the 6-day storage were fastest for beef, while such decline and increase during the entire storage period were slowest for chicken B. Overall trends of natural meat flavor and CBD intensity changes for chicken T appeared more like those for the red meats than chicken B. It was concluded that, while flavor deterioration can occur in cooked-stored meats from all the species, quantitative or the magnitude of differences between species would depend on muscle types and sensory terms/method used.