SB/T 10923—2012掺假肉类DNA检测方法的改进与评价

目的 我们对SB/T 10923—2012《肉及肉制品中动物源性成分的测定 实时荧光PCR法》检测方法进行改进,评价其应用于掺假肉DNA鉴定的价值。方法 评价改进后方法的特异性和掺假比例检测能力,并用7份预包装生鲜冷冻肉制品考察实际检验效能。结果 改进后的方法在Cq阈值为30时,能够有效区分20 ng/反应的特异模板,最低可检出0.5%（w/w）的掺假比例。对7份样品共进行24个肉源成分检测,与标签符合率为95.8%（23/24）。有1种鸭源性成分未检出,可能和混样不均匀有关。结论 改进后的方法可快速、特异、简便地用于掺假肉常规检测。     

基于DNA条形码技术常见肉类掺假鉴别技术的研究

根据市场上常见的肉类掺假情况,本研究通过提取生鲜牛肉、羊肉、猪肉和鸭肉基因组DNA,按一定比例进行预混合,构建牛肉掺猪肉、羊肉掺猪肉、牛肉掺鸭肉和羊肉掺鸭肉4种掺假模型。通过引物COI-1和COI-2进行PCR扩增和测序比对,建立基于COI基因的动物源性食品的掺假判别方法。根据实验所得纯肉DNA提取率T实现DNA水平到肉水平掺假比例的换算。在肉的掺假水平上,引物COI-2检测效果较好,对牛-猪、羊-猪、牛-鸭和羊-鸭模型掺假物的检出限分别为5%、8%、1%和4%。对采集的28个批次的肉制品进行检测,结果表明:28个样品中89%的样品与产品标签标识的成分相符。建立的基于DNA条形码技术的检测方法可作为一种简单、快速、有效的分子鉴定技术,可以直接应用于研究物源性食品的种类和掺假鉴定。     

利用可视基因膜芯片技术与实时荧光定量聚合酶链式反应技术分析肉制品中动物源性成分

为调查了解肉制品中动物源性成分,以帮助判别掺假情况,应用可视基因膜芯片检测技术对市售的肉松、香肠、肉卷、预制调理肉、肉干及肉脯等23份样品动物源性成分进行筛查分析,同时,针对筛查结果采用实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）法进一步确证。结果表明：可视基因膜芯片检测法与实时荧光定量PCR法检测结果一致,提高了未知样品的筛查效率;在本次随机分析的样品中,动物源性成分检测结果与标签标示不一致的情况占比高达21.7%,肉制品掺假虚标情况不容忽视。     

多重荧光PCR鉴别羊肉掺假

目的建立可同时检测掺入羊肉中的猪、马、牛、鸭成分的多重荧光PCR检测体系。方法配制掺入猪、马、牛、鸭源性成分的模拟羊肉掺假样品,掺假比例17%~45%。筛选特异性强、敏感度高的猪、马、牛、鸭物种特异性基因,优化反应体系,建立了针对猪、牛、马、鸭两两配对的两重荧光PCR检测体系和其中3个任意组合的三重荧光PCR检测体系。结果模拟羊肉掺假样品两重荧光PCR检测结果显示,包括单个成分掺入比例低至7%的样品,其检测准确率为100%。而三重荧光PCR检测同时检测猪、牛、马、鸭四种肉品中的3种,其中猪肉、牛肉和马肉的掺假比例均为8%~15%;鸭牛肉掺假比例为5%~10%。三重荧光PCR检测结果显示,所有掺假样品中的各种掺假组分都被准确检出。结论本研究建立的多重荧光PCR检测体系能准确检出配制的模拟掺假羊肉制品中的猪、马、牛、鸭成分,可进一步研究以应用于市场肉制品及加工肉制品掺假的检测。     

实时荧光PCR定量测定肉制品中羊源性成分

为准确定量肉制品中羊源性成分的含量,以羊单拷贝基因为扩增靶基因,设计合成羊源的引物、探针,制备羊源和脊椎动物通用基因的重组质粒以构建标准曲线,通过方程算出各自的拷贝数,根据拷贝数的比值计算样品中羊肉源成分占总肉成分的百分比含量,建立荧光定量PCR检测体系,并对已知目标肉含量的混合肉样进行定量检测。结果显示,该方法具有良好的特异性和灵敏度,最低检测到羊的DNA含量为0.02 ng/μL。对已知目标肉含量的混合肉样进行定量检测发现,羊肉掺入量为90%、50%时所得结果相对准确,相对标准偏差分别为5.69%和10.82%, 且不受外源物种的干扰。本试验建立的定量PCR方法能够准确检测出不同样品中羊肉的含量,并发现存在掺假现象。     

食品中羊肉源性成分微滴数字PCR定量方法的建立

为了对肉制品中羊肉源性成分进行准确定量,该研究采用微滴数字PCR(dd PCR)技术对羊肉的单拷贝基因进行定量检测,根据基因拷贝数建立肉制品中羊肉源性成分dd PCR的定量检测方法。结果表明,通过向样品中添加牛肉作为内标,建立了基于DNA拷贝数与样品质量间线性关系对羊肉源性成分进行dd PCR内标定量的方法,实现了从靶基因拷贝数到样品质量间的一步转化。该方法在检测出0. 01%的羊肉源性成分时,检测结果达0. 12 copies/μL,能够对含量5%以上的羊肉源性成分进行准确定量。通过对已知成分的混合样品和市售样品进行检测,显示该方法能够准确检出不同样品中羊肉源性成分含量。因此,该方法在肉及肉制品中羊肉源性成分检测和掺假鉴别方面具有较大应用潜力。     

4种动物源性成分多重real-time PCR检测方法的建立及其在驴肉制品检测中的应用

建立一种同时快速检测驴、马、猪及鸭源性成分的四重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以4种源性成分的Nad5、ATpase6、ATP8、cytb基因为靶基因设计特异性引物和Taq Man探针,以18S r RNA基因为内参基因,建立多重real-time PCR方法,并对该方法进行方法学验证,同时对不同掺入比例模拟样品、不同加工工艺模拟样品和实际驴肉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。当Ct值≤35.0时,方法对16种非目标源性具有良好特异性;灵敏度可检测到质量浓度为2×10^-4 ng/μL的模板DNA;对生肉的检出限为肉含量的0.001%,对熟肉制品的检出限为肉含量的0.01%;对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可用于肉及肉制品中常见掺假源性成分的检测。     

肉制品中狗、狐、貂源性成分DNA检测试剂盒的制备

为快速检测肉制品中狗、狐、貂3 种常见犬型总科动物源性DNA成分,应用分子生物学技术分析种属基因组序列差异,建立并优化多重聚合酶链式反应检测体系,开发快速检测试剂盒。结果表明：快速检测试剂盒特异性良好,与常见肉类动物（猪、牛、羊、马、驴、鸡、鸭）源性DNA成分皆无交叉反应;灵敏度高,3 种靶标样品DNA质量浓度同时降至10－4 ng/μL,仍可准确检测;阳性条带经切胶回收、克隆转化、测序比对,与GenBank已登记的狗（MH105046.1）、狐（LT560065.1）、貂物种（KU145464.1）相似性为100%;对30 份模拟混合肉样进行鉴别检测,检测结果与预设混合情况完全相符;50 份市售样品中,狗肉样品中3 份出现掺假现象。     

DNA条形码COI序列在常见肉类鉴别中的应用研究

为了对常见的4种肉类及相关肉制品进行掺假鉴定,判别与产品标签是否相符,本研究以COI基因为靶基因,建立了4种动物源性食品DNA条形码鉴别技术。分别提取牛、羊、猪、鸭四大物种的基因组DNA为模板,以其COI基因的保守序列区设计6对通用引物,结合文献报道及数据库提供的7对通用引物进行PCR扩增,并将测序结果提交Gen Bank数据库Blast比对,评价不同DNA条形码的检测鉴别能力。筛选出COI-A为最优序列,在4个物种中扩增效率100%。对抽检的20个批次的肉加工品样品进行检测,鉴定结果约有90%的样品与产品标签标示的成分相符。其中1个批次的牛丸制品因肉类成分含量低未扩增成功,1个批次的牛丸制品检出鸭源成分,判定掺假。DNA条形码技术快速有效,本研究筛选的COI-A序列可直接用于牛、羊、猪、鸭及其肉制品的鉴定,并为其它常见动物源性食品的种类鉴定提供一定参考依据。     

肉类掺假的分子生物学检测

针对Gene Bank中公布的牛羊猪鸡鸭线粒体细胞色素氧化酶亚基基因的特异性序列设计引物,建立了肉样的5种常见牛羊猪鸡鸭动物源性成分检测的PCR电泳法体系。采用大连宝生物的猪源牛源羊源鸡源性成分检测试剂盒和广州迪奥生物的鸭源性成分检测试剂盒,利用实时荧光PCR检测体系对河南省范围内几个大型超市的118批次牛肉样品的5种即猪牛羊鸡鸭动物源性成分进行了检测。结果显示:牛源性成分检出117批次检出率99.15%,猪源性成分检出10批次检出率8.47%,鸡源性成分检出7批次检出率5.93%,鸭源性成分检出1批次检出率0.85%,没有检出羊源性成分,其中熟肉制品掺杂其他源性成分的比例较高。与配料表对比掺假使假样品共9批次,总掺假率为7.63%。     

液相色谱-串联质谱法测定肉品中氯噻啉药物残留

采用液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry,LC-MS/MS）法测定肉 品中氯噻啉药物残留。肉样经过酸化乙腈提取后,采用N-丙基乙二胺和C18吸附剂净化,然后在BEH-C18柱 （2.1 mm×50 mm,1.7 μm）上用乙腈和体积分数为0.1%的甲酸水溶液为流动相进行梯度洗脱,流速0.30 mL/min。 样品经电喷雾离子源正离子模式电离后,在多反应监测模式下定性及定量。结果表明：该方法在10～400 ng/mL范 围内线性关系良好,方法检出限为5.0 μg/kg,回收率为91.41%～93.51%,相对标准偏差在2.4%～3.2%之间。该方 法结合了QuEChERS（quick,easy,cheap,effective,rugged,safty）和LC-MS/MS的优点,操作简便、分析速度 快、灵敏度高、重现性好,可以用于肉品中氯噻啉残留的检测。     

COI序列应用于羊肉掺伪非定向筛查技术的研究

本项目研究了应用COI序列对羊肉中掺入的其他动物源材料进行非定向筛查,并对山羊肉及绵羊肉进行分辨。利用了DNA条形码技术对六个羊品种(系)的线粒体基因组细胞色素酶基因片段进行引物筛选,并利用筛选出的引物对七种动物源样本的COI片段进行扩增,以确定所筛选出的引物是否能将羊肉与其他动物源样品进行有效区分。以LCOI490/HCO2198、F1-1/R1-1、ITS3/ITS4为引物,通过对36份血液样本(山羊3个品种,绵羊3个品种)的基因组DNA进行PCR扩增,其中以F1-1/R1-1为引物得到的PCR产物电泳检测条带清晰,结果重现性好;利用F1-1/R1-1引物对鸡肉、猪肉、驴肉、牛肉、莱芜黑山羊肉、洼地绵羊肉和鸭肉等七种样本的COI片段进行扩增,并对扩增片段进行测序,山羊、绵羊之间的序列同源性仅为36.62%,其他各品种间的序列差异也很明显。结果表明:以F1-1/R1-1为专用引物可以有效区分羊肉与其他动物源样品,并能够对山羊肉及绵羊肉样品进行有效区别。     

ELISA法与LC-MS/MS法测定动物组织中克仑特罗残留

目的:比较酶联免疫法(ELISA)和液相色谱-串联质谱法(LC-MS/MS)测定动物组织中克仑特罗残留量的准确度、精密度及检测限。方法:样品经过处理后,分别采用ELISA法和LC-MS/MS法进行测定;用ELISA法对组织样品进行初筛,测出阳性样品利用LC-MS/MS法进行确证。结果:应用ELISA法测定样品猪肉样品中克仑特罗的回收率为67.0%～99.6%,批内变异系数3.4%～8.7%,批间变异系数6.1%～9.6%,灵敏度为0.025μg/L,最低检测限0.025μg/kg;LC-MS/MS检测方法回收率88.62%～111.43%,批内变异系数4.4%～7.4%,最低检测限0.5μg/kg;用ELISA法对50份猪肉和50份猪肝样品进行检测,筛选出3个阳性样品,经LC-MS/MS确证为阳性,两种方法检测结果一致。结论:ELISA法灵敏度和准确度较高,样品处理方法简单,成本低,适合组织中克仑特罗残留大规模筛查;LC-MS/MS准确度高,适合于阳性样品精确定量。     

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.     

Duplex PCR approach for the detection and quantification of donkey,horse and mule in raw and heat‐processed meat products

Donkey‐related products have been paid more attention for their high nutritional value to human beings. Due to donkey resource scarcity, coupled with gradually increasing market demand, adulterated donkey meat products with other low‐cost animal meat, especially with the similar species horse and mule, are often found in market. Therefore, detection of species fraud in donkey meat products is important for consumer protection and food industries. In this study, a simple and highly specific duplex PCR method, based on the simultaneous amplification of fragments of the mitochondrial ATP synthase subunit 8/6 and ND2, was developed and optimised for the identification of horse, donkey and mule species in raw and heat‐processed meat products. To the best of our knowledge, it was the first time for this strategy applied to these three genetically related animal meat products differentiation to date. The duplex PCR generated a 153‐bp and 83‐bp amplification products for horse and donkey, respectively. While for mule, both of the two length amplification products are appeared on the agarose gel. Target meat species could be detected at a level of 1%, and the results indicated that the duplex PCR assay could be used in the authentification of donkey‐related products with high specificity, cost‐effectiveness and simplicity.     

Identification of Ostrich Meat by Restriction Fragment Length Polymorphism (RFLP) Analysis of cytochrome b Gene

ABSTRACT: To enforce labeling regulations in the authentication of ostrich meat, it might be of importance to evaluate a method to identify the ostrich meat. A restriction site of the polymerase chain reaction (PCR) product has been used for the specific identification of ostrich meat. In the present study the part of the gene encoding cytochrome b was amplified and sequenced. The digestion of the PCR products using specific estriction enzymes Hae III, Hinf I, Rsa I, and Tru 91 were used to yield specific restriction profiles that allowed a direct identification of ostrich meat in raw and heat-treated samples from meat of other food animal species.     

一对同时鉴定8种动物源性成分的通用引物的制备及应用

肉类真假鉴定是食品检测工作的内容之一,目前已有多种基于PCR的肉类鉴定方法,但是鉴定种类和效率受限。本研究设计了一对基于普通PCR技术可同时鉴定8种动物源性成分的通用引物并建立了鉴定方法。该引物以线粒体DNA为靶标,利用扩增产物中不同物种间的插入缺失多态性片段大小即可鉴定山羊、绵羊、鹿、水牛、牛、牦牛、猪和骆驼8个物种,扩增后分别得到728 bp、704 bp、504 bp、453 bp、448 bp、431 bp、396 bp和326 bp的片段,每种PCR产物经SspI酶切后产生数量和大小不同的片段,可以进一步清晰鉴别8个物种。引物特异性测试表明和其他常见肉类动物DNA无交叉反应,DNA检测最低限度在0.01~0.05 ng。应用本方法对40份市场肉类及产品的检测表明,羊肉串、羊肉卷以及特色畜产品如驼肉、鹿肉和驴肉存在较多的掺假行为。与其他现有PCR检测方法相比,该方法具有简便易行和高通量的优点,可以作为肉类掺假筛选检测的常规方法。     

PCR技术检测动物源性食品中沙门菌的应用研究

为快速准确检测动物源性食品中的沙门菌,将建立并优化的沙门菌PCR检测试剂盒应用于动物源性食品的检测,并于国标法进行了比较.对上海地区238份鸡蛋、685份原料牛奶、283份猪肉、314份牛肉和58份虾仁样品的沙门菌检测表明,PCR法的敏感性和特异性均为100%,与国标法的符合率亦为100%,且检测时间仅为2 d,较国标法大为缩短.该方法可用于动物源性食品中沙门菌的检测,并具有快速、特异、敏感等优点。     

Development and application of highly specific PCR for detection of chicken (Gallus gallus) meat adulteration

In order to prevent fraud in the sale and strengthen quality assurance, authentic identification of chicken meat is essential. In the present investigation, a chicken (Gallus gallus)-specific polymerase chain reaction (PCR) was developed for the unambiguous identification of chicken meat. The PCR assay employs pair of primers designed against chicken nuclear 5-aminolevulinate (ALA) synthase gene. Highly chicken-specific diagnostic amplicon of 288 bp was established upon PCR and was evident in all the nine breeds/strains of chicken species. Sensitivity of PCR in detecting chicken meat adulteration was established to be at 0.1 % in the foreign meat matrix, while limit of detection (LOD) of chicken DNA was 10 pg. Suitability of the developed chicken-specific PCR was validated and confirmed in raw, cooked/heat treated (60, 80, 100, and 121 °C), and micro-oven cooked meat samples. Possibility of cross-amplification of adulterating DNA was excluded by cross-checking the developed PCR assay with several animal and avian species. The PCR assay developed in this study is highly promising for applications involving circumstances that require authentic identification of chicken meat.     

Simultaneous determination of residues of trichlorfon and dichlorvos in animal tissues by LC-MS/MS

Trichlorfon is a thermally unstable insecticide that can be easily decomposed to dichlorvos at high temperatures. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of the residues of trichlorfon and dichlorvos in animal tissues. Trichlorfon and dichlorvos in animal tissues were extracted with dichloromethane, homogenized/shaken, concentrated, and determined by LC-MS/MS. The two insecticides can be effectively separated and individually determined. The limits of detection (LODs) of trichlorfon and dichlorvos were 0.04 and 0.07 µg kg^?1, respectively; the limits of quantification (LOQs) of the two insecticides were both 5 µg kg^?1. The average recoveries for three spiked blank samples at 10, 20 and 40 µg kg^?1 were in the range 85–106%, with relative standard deviations (RSDs) below 10.6%, which falls in a reasonable range for the analysis of the two insecticides in animal-derived foods. The method is fit-for-purpose for the simultaneous determination of residues of trichlorfon and dichlorvos in animal tissues.