Polymerase chain reaction assay for identification of chicken in meat and meat products

The aim of this study was to develop polymerase chain reaction (PCR) assay for specific detection of chicken meat using designed primer pair based on mitochondrial D-loop gene for amplification of 442 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The PCR result was further verified by restriction digestion with HaeIII and Sau3AI enzymes for specific cutting site in amplified DNA fragments. The specificity of assay was cross tested with DNA of cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail, where amplification was observed only in chicken without cross reactivity with red meat species. However positive reaction was also observed in quail and turkey. In this study, no adverse effects of cooking and autoclaving were found on amplification of chicken DNA fragments. Thus, the detection limits was found to be less than 1% in admixed meat and meat products. The developed assay was found specific and sensitive for rapid identification of admixed chicken meat and meat products processed under different manufacturing conditions.     

Authentication of carabeef (water buffalo, <Emphasis Type="Italic">Bubalus bubalis</Emphasis>) using highly specific polymerase chain reaction

In order to ensure consumer satisfaction and fraud detection, correct identification of meat animal species becomes significant. Buffalo being one of the major meat animal species in Asia, a species-specific polymerase chain reaction (PCR) was developed for the accurate identification of carabeef (water buffalo, Bubalus bubalis) targeting mitochondrial D-loop region. Unique diagnostic PCR developed in this study employed novel primers to yield a 534-bp buffalo-specific PCR product, and chances of cross-amplification were excluded by including as many as 25 animal species. Applicability of PCR was established in raw, cooked (60, 80 and 100 °C for 30 min), autoclaved (121 °C for 30 min) and microoven-processed meats with a sensitivity of detection of 0.1% adulteration (10 pg bubaline DNA). Keeping in view adulteration, socio-economic, religious, quality assurance, forensic and legal issues, the novel buffalo-specific PCR developed in this study was found highly promising in authenticating buffalo meat, ensuring consumer satisfaction and labeling process.     

基于便携式荧光定量PCR仪定量检测驴肉中掺假鸭肉

为建立快速方便的驴肉制品分子鉴定方法,本文以驴肉和常见的掺假肉类(鸭肉)为研究对象,筛选特异性引物和TaqMan探针,利用便携式Mini8 Plus实时荧光定量PCR仪进行灵敏度和特异性实验,通过绘制扩增标准曲线及确定驴肉和鸭肉的质量与DNA比值常数,对不同掺入比例(加入定量的鸭肉制成含量分别为20%、40%、60%、80%)的模拟样品和实际驴肉样品进行检测。结果显示,该方法对驴、鸭肉均具有良好的特异性,可以与马、猪、山羊、梅花鹿、牛、鸡、狗肉明显区分;对驴源性DNA成分的检出限为0.01 ng/μL,鸭源性DNA成分的检出限为0.1 ng/μL,对驴肉与鸭肉混合物中鸭肉成分的灵敏度为0.1%(w/w);所建立的标准曲线线性关系良好,驴肉DNA扩增标准曲线:y=-3.584x+27.003,R²=0.9982;鸭肉DNA扩增标准曲线:y=-3.538x+30.907,R²=0.9991;采用已建立的方法对35份驴肉样本进行市场试点调查,发现6份(17.1%)驴肉样本中含有鸭肉成分。以上研究结果说明,该实时荧光定量PCR方法可用于驴肉产品中其他...     

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。     

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...     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

基于DNA检测的 肉制品鉴伪技术研究进展

在利益的驱动下,肉制品掺假行为屡禁不止,因此肉制品鉴伪技术逐步发展。本文综述了近五年来常见的基于DNA检测的肉制品鉴伪技术,包括经典的PCR、qPCR、DNA条码技术,新兴技术如下一代测序技术、极灵敏的HRM、用于基因诊断的LAMP,经典DNA标记如微卫星技术、RAPD、AFLP、RFLP、序列特征扩增区域、单核苷酸多态性等,为肉制品掺假鉴伪等相关研究提供借鉴。     

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。     

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。     

肉制品异源基因检测技术研究进展

近年来,食品掺假逐渐成为消费者关注的重要食品安全问题之一。由于利益的驱使,在肉制品行业异 源肉质掺假现象尤其严重。目前,用于肉制品异源基因检测的技术包括普通聚合酶链式反应（polymerase chain reaction,PCR）技术、DNA指纹技术、实时荧光定量PCR技术、微滴式数字PCR技术、DNA条形码技术等。本文 综述了肉制品中异源基因检测技术的研究进展,并对每种方法的优缺点和发展趋势予以讨论。     

核酸检测技术检测肉类种源研究进展

肉及肉制品是人类重要的食物来源, 为人体提供必要的营养素。但是以经济为目的的肉制品掺假, 是食品安全中屡禁不止的全球性问题。快速、准确、有效的检测技术是有效监督肉类掺假的关键。本文综述了核酸检测技术中热循环扩增技术(如普通PCR、实时PCR、多重PCR)和等温扩增技术[如环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术、重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术、滚环扩增(rolling circle amplification, RCA)技术、交叉引物等温扩增(cross prime amplification, CPA)技术等]的原理及在肉类种源鉴别中的应用。提出梯型熔解温度等温扩增(ladder-shape melting temperature isothermal amplification, LMTIA)技术, 以期推进核酸检测技术的研究及在肉制品领域的应用。在肉类种源的检测中, 实时荧光定量PCR技术、跨越式滚环等温扩增(saltatory rolling circle amplification, SRCA)技术等均能检出0.01%的掺伪, 可用于定量检测, 表明这些核酸技术在肉类种源检测中有很好的应用前景。     

Establishment and application of a polymerase chain reaction for the identification of beef

A polymerase chain reaction (PCR) based method for the identification of beef by amplification of bovine 1.709 satellite DNA was established. The method not only was able to amplify raw beef DNA, but also cooked or autoclaved meat DNA. The sequence selected for amplification consisted of a 218 bp DNA fragment lying in the 1.709 satellite DNA of bovine. A pair of synthetic oligonucleotides flanking this sequence were used as printers, and genomic DNA extracted from beef samples employed as templates. Each batch of reaction mix contained Taq DNA polymerase, a buffer component, deoxynucleotide triphosphates, genomic DNA template and a pair of bovine oligodeoxynucleotide primers in a final volume of 50 μl. The amplification of bovine DNA was performed by using 33 cycles of denaturation at 94°C (40 s), annealing at 53.5°C (50 s) and extension at 72°C (60 s), with a 7 min extension at 72°C in the last cycles. The amplified products were subjected to rapid electrophoresis in 3% agarose gel and visualized under ultraviolet illumination after ethidium bromide staining. A Hae Ш restriction endonuclease test was done to verify the specificity of the PCR amplification, and the expected DNA fragments were produced. The specificity test demonstrated that this method was positive for bovine, buffalo and yak meat DNA, but negative for equine, sheep, goat, camel, swine, deer and mouse meat DNA, etc. At least 33.6 fg of DNA from raw beef samples and 0.32 pg of DNA from cooked or autoclaved beef samples were detected, respectively, by PCR. We tested 103 beef samples by PCR and obtained 100% correct identification. The method needed only 6 h for detection of meat products of all kinds. The results showed that the PCR method was sensitive, specific, convenient and rapid, so it may be suitable for rapid identification of beef.     

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.     

肉及肉制品中沙门氏菌活细胞的PCR检测研究

将荧光染料叠氮溴化丙锭(propidium monoazide,PMA)与普通聚合酶链式反应(polymerase chain reaction,PCR)结合,通过对PMA的曝光时间、浓度进行优化,确定PMA-PCR区别死活细胞的最佳条件,并制作活细胞定量标准曲线,建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min;不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL;完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理,含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测,最小检测限为20CFU/PCR。而且,经研究发现在20～2×105CFU/PCR范围内,电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品,利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌,经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。     

肉制品中羊源性成分的定性和定量检测

通过检测肉制品中DNA的来源进行动物源性检测是打击鲜肉及加工肉制品制假掺假的重要技术手段。依据GB/T 25165—2010《明胶中牛、羊、猪源性成分定性检测方法 实时荧光PCR法》合成用于TaqMan 实时荧光聚合酶链式反应的引物和探针,利用鲜肉及加工肉制品进行羊源性成分定性和定量检测方法研究。首先利用12 种不同动物鲜肉组织的DNA检测方法的特异性,然后以羊源DNA梯度稀释液为模板进行灵敏度实验,最后在加工肉制品中检测方法的适用性和定量检测能力。结果表明：此方法具有羊源性成分检测特异性,对其他动物来源的鲜肉DNA均无扩增信号,可以检出羊肉和猪肉混合样品中0.1%的羊肉;方法灵敏度高,可以检出10 pg的羊源DNA;方法适用性广,可以对加工肉制品（羊肉干）进行羊源性成分的定性和定量检测。     

微滴式数字PCR对肉制品中羊肉和猪肉定量分析

为了实现肉制品中羊肉和猪肉的量化研究,本文应用微滴式数字PCR对肉制品进行定量检测。通过在看家基因上设计单拷贝特异性引物,能更好的鉴别是人为因素的掺假还是在加工过程中无意的带入。根据dd PCR的结果显示,在一定范围肉质量和DNA的浓度,DNA浓度和DNA拷贝数(C)成现一定的线性关系。通过人为掺假及市售样品检测以验证此方法。以DNA浓度作为中间换算值,得到肉质量和DNA拷贝数之间的换算关系M_羊=0.12C-10.6 M_猪=0.07C+4。通过对已知掺假比例的肉样及市售样品进行检测,能够较好的得到掺假肉的质量。目前市场上存在一定的掺假现象且该检测方法具有一定市场应用前景,本文建立的微滴式数字PCR在羊肉和猪肉及其制品中的掺假检测具有较强的应用潜力,对目前混乱的肉制品掺假的市场监管提供了科学的依据。     

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.     

可视化LAMP检测常见肉制品中猪肉成分

根据GenBank中公布的猪线粒体色素细胞b基因序列设计6 条特异性环介导等温扩增（loop-mediated isothermal amplification,LAMP）引物,通过简化线粒体DNA提取步骤,优化反应体系,以常见的牛、羊、鸡、鸭、马、驴肉为阴性对照验证该方法的特异性,掺假率梯度稀释以验证该LAMP法的最低检测限,建立常见肉制品中猪肉成分的可视化LAMP检测方法。结果表明：该方法提取目的基因操作简单、稳定,该可视化LAMP检测法以4-(2-吡啶偶氮)-间苯二酚钠盐为指示剂,能准确检测出常见肉类中猪肉是否掺杂,检测结果肉眼可见,灵敏度为10 pg/μL。     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

Biochip Technology for the Detection of Animal Species in Meat Products

The verification of declared components in meat products is an essential task of food control agencies worldwide. To date, the ELISA and species-specific polymerase chain reaction (PCR) are two commonly applied analytical tools employed by many authorized food control laboratories. These trusted methods however do not allow the simultaneous detection of all the animal species present in a meat sample. Additionally, detection of undeclared components resulting from inadvertent contamination or deliberate adulteration of the meat products requires additional processing of the samples, resulting in increased expenditure. The use of DNA biochip analysis that allows simultaneous processing of many meat products, while concomitantly generating results for the detection of all animal species present in the meat products is thus highly desirable. In this work, two commercially available animal chip detection systems (CarnoCheck Test Kit and MEAT^species LCD Array) are compared in terms of sensitivity, robustness, reproducibility, and ease of handling. The two animal species differentiation biochip methods compared well in efficiency and could simultaneously detect from eight to 14 animal species in the meat products. Detection limits were found to be in the range of 0.1% to 0.5% in meat admixtures, with good reproducibility of results. More than 70 commercially available meat samples were analyzed in this work, with the results validated against traditional PCR methodology. Both biochip methods performed well and could be implemented for routine use in any food control agency.