A review of current PCR-based methodologies for the authentication of meats from game animal species

The authenticity of food is currently a major issue for researchers, consumers, industries and policy markers at all levels of the production process. Particularly in the meat industry, products from game animals are susceptible targets for fraudulent labeling due to the economic profit that results from selling cheaper meat as meat from more profitable and desirable species. A part from meat species adulteration, illegal poaching of endangered game species may take place contributing to threat of wildlife populations. These reasons have encouraged the development of methods to ensure fair trade and labeling of game meats from production level to consumer use of end products. In the last years, full attention has been turning towards implementation of molecular genetic approaches for meat species identification because of their high sensitivity and specificity, as well as rapid processing time and low cost. This work presents an overview of the main PCR-based techniques applied to date to verify the authenticity of meat and meat products from game species.     

Multiplex PCR in Species Authentication: Probability and Prospects—A Review

Food forgery is one of the most articulated socio-economic concerns which contributed to increase people’s awareness on what they eat and how and where it is produced. Consumers are anxious about the consequences of food falsification on their choices, religious rituals, health, and hard-earned fortunes. The recent scandals of horse and rat meats in Europe and China have given us a brainstorming apprehension on the detection, differentiation, and identification of meat products. To restore consumers’ trust and protect wildlife in natural habitats, researchers and policy-making and policy-implementing authorities have massively monitored all steps in the production of foods and food materials. Analytical approaches based on lipids, proteins, and DNA have been proposed for the authentication of meat species under pure and complex matrices. However, protein and lipid-based methods are less effective since the target biomarkers could be modified throughout the processing treatments. On the other hand, DNA-based species identification schemes have gained wider acceptance and reliability because of the superior stability and universality of DNA in all tissues and cells. We systematically presented here major species detection schemes with special emphasis on multiplex polymerase chain reaction (PCR) of both end-point and real-time platforms. We believe this short but comprehensive review would serve as a reference guide for the developers and users of multiplex PCR and others DNA-based techniques.     

Authentication technologies using DNA-based approaches for meats and halal meats determination

Food authenticity and safety are major concerns for researchers, consumers, and particularly the meat industry. Meat products are targets for species substitution and adulteration due to their market value. Presently, the demand for halal products is witnessing a substantial increase. Therefore, it is essential to use appropriate science-based methods for determining the species origin of halal meat. DNA barcoding is a useful technique for the molecular identification of biological specimens, and raw and processed foods. The potential of using DNA barcoding is increasingly applied as an authentication tool for halal animal and meat products. Our review will bring together all DNA-based techniques that have been developed for the authenticity of meat derived from halal and non-halal animals and also their derivatives. Additionally, the present paper will highlight the possibility of using the DNA barcoding approach for halal meat authenticity.     

基于核酸分子学方法的肉类成分鉴别技术研究进展

近年来,肉类掺假问题频繁发生。基于核酸的分子生物学肉类成分鉴别技术已成为研究热点,其具有灵敏度高、特异性强、检测时间短以及成本低的优点。本文综述了基于核酸分子学的肉类成分种属鉴别技术在肉类掺假检验中的应用,着重于量化各种方法的检测限,并重点对实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)和数字PCR技术在动物成分鉴别定量分析的研究现状与前景做介绍。探讨不同来源的靶基因(核DNA和线粒体DNA)在动物成分鉴别中,定性和定量检测灵敏度与特异性的区别。     

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.     

Identification of hare meat by a species-specific marker of mitochondrial origin

Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat.     

基于肽生物标志物技术鉴别肉品的真实性研究进展

基于DNA和蛋白质的方法已广泛应用于肉制品的真实性鉴定,但肉制品经加工处理后,DNA和蛋白质很容易发生降解和变性,而蛋白质的一级结构(氨基酸序列)则足够稳定。伴随着蛋白质组学的发展,以肽生物标志物为基础的鉴定方法逐渐应用于肉制品的真实性分析。本文将从肉中肽生物标志物的分离鉴定方法、肽生物标志物在鉴定不同肉制品及肉制品中外源蛋白的应用三个方面进行归纳和总结,并对肽生物标志物在鉴定肉品真实性应用中的发展方向进行展望,以期为食品溯源提供更丰富的技术手段。     

Hamburger meat identification by dot-ELISA

The use of low cost meats to adulterate meats and meat products has been reported. Appropriate methods of analysis then are needed in order to detect this practice. The dot-ELISA method was used to identify the meat of different animal species and to detect adulteration of hamburgers. Antisera to bovine, chicken, swine and horse albumin were produced and they could detect the meat extract of the homologous species at concentrations as low as 0.6%. Thus, the anti-albumin antisera could identify bovine, chicken, swine and horse meat with adequate specificity and sensitivity both in isolation and when added to hamburger. Commercial samples of bovine, chicken and swine hamburgers showed no adulteration with bovine, chicken, swine or horse meats. Our expectation of hamburger adulteration was not confirmed.     

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。     

食品中动物源性成分的多重PCR快速检测

目的利用多重PCR技术对市售的多种食品进行检测,确定动物源性成分的种类,进行掺假及真伪鉴别。方法对鸡、猪、牛、兔、绵羊、山羊、马、鹿8种动物源性产品进行DNA提取,根据不同动物线粒体DNA设计特异性引物,根据脊椎动物细胞色素b基因mt DNA序列设计通用引物作为内源参照,对8种动物源性产品的DNA进行多重PCR扩增,同时对多种成分进行鉴定。结果琼脂糖凝胶电泳表明,此方法能同时扩增出8种动物的特异性条带,检测灵敏度达到0.01%。结论所建立的鉴定多种动物源性成分的新方法,操作简便、快速准确,可为各检验机构对食品进行检测提供方法指导。     

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 meat species by TaqMan-based real-time PCR assay

In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.     

Multiplex real-time PCR for the detection and quantification of DNA from beef,pork, horse and sheep

Meat products are often composed of more than one meat species. A quantitative multiplex PCR was developed to determine the proportion of meat fractions of beef, pork, horse and sheep. The precision and accuracy were investigated by dilutions of DNA from all four species and examining different meat products from the market. Application of this tetraplex quantitative real-time PCR system will enable official food control and production control laboratories to efficiently investigate the composition of meat products.     

Determination of Sex Origin of Meat and Meat Products on the DNA Basis: A Review

Sex determination of domestic animal's meat is of potential value in meat authentication and quality control studies. Methods aiming at determining the sex origin of meat may be based either on the analysis of hormone or on the analysis of nucleic acids. At the present time, sex determination of meat and meat products based on hormone analysis employ gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS), and enzyme-linked immunosorbent assay (ELISA). Most of the hormone-based methods proved to be highly specific and sensitive but were not performed on a regular basis for meat sexing due to the technical limitations or the expensive equipments required. On the other hand, the most common methodology to determine the sex of meat is unquestionably traditional polymerase chain reaction (PCR) that involves gel electrophoresis of DNA amplicons. This review is intended to provide an overview of the DNA-based methods for sex determination of meat and meat products.     

稳定同位素、矿质元素及可见-近红外光谱技术在禽制品真实性鉴别中的应用研究进展

禽制品在食品消费中占据很大比例,近年来,禽制品掺假、造假等事件频发,禽制品真实性鉴别成为研究热点.随着掺假造假手段的多样化,传统的感官鉴别方法已经难以满足禽制品真实性鉴别的需要.近年来,现代分析仪器和技术发展迅速,现代分析方法在禽制品真实性检测中展现出优势.本文重点阐述稳定同位素技术、矿质元素指纹技术及可见-近红外光谱...     

Species-specific multiplex real-time PCR assay for identification of deer and common domestic animals

A multiplex real-time PCR method for discriminating deer and common domestic species, including cattle, goat, horse, donkey, pig, and chicken was developed. Species-specific primer pairs were designed and used to produce different size DNA fragments with diverse melting temperature (T _m ) values. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. Multiplex real-time PCR analysis was performed using combined primers, yielding distinct melting curve profiles for each species. The sensitivity limit of the multiplex PCR method was evaluated. Trace DNA of other species in deer DNA could be identified. Common deer products, including blood, meat, and antler were tested using this multiplex PCR method and different species of deer and domestic animals were identified. The rapid multiplex real-time PCR assay described herein is a specific, sensitive, and reliable method for high-throughput authentication of deer and domestic animal products.     

Authenticity Determination of Meat and Meat Products on the Protein and DNA Basis

Adulterated food can be defined as food incompatible with the declaration of the seller. In the case of meat and meat articles, adulterations refer not only to the replacement of ingredients but also to inappropriate information concerning the origin of raw materials. Methods aiming at investigating meat and meat product authenticity may be based either on the analysis of protein composition or on the analysis of nucleic acids. At the present time, meat and meat product authenticity investigations based on protein analysis employ electrophoretic, enzymic, and chromatographic methods, sometimes supported by the mass spectrometry technique. On the other hand, species identification is often based on polymerase chain reaction (PCR). Biochips present a promising technology.     

PCR技术在肉类掺假检验中的应用进展

当前,对研究者、消费者、食品工业和政策制定者等各个方面来说,食品的真伪都是一个热点问题,尤其是肉类工业。PCR技术具有特异性强、敏感性高、操作简便、快速高效等特点,在肉类掺假检测方面具有巨大的应用价值。本文介绍了目前肉制品鉴定的方法,包括蛋白和核酸两个层次,用于肉制品鉴定的各种目的基因的选择。回顾了PCR技术在国内外肉类产品掺假鉴定中的重要应用。指出了PCR技术在肉制品鉴定中的不足,与各种新技术(基因芯片、蛋白质芯片等)有机结合将是以后的研究方向。     

肉制品鉴别的分子生物学方法研究进展

世界各国每年的肉类掺假事件频发。多种基于核酸、蛋白质和脂质的技术被开发出来并实际应用于检测肉类真伪。近二十年来,随着分子生物学检测技术的迅猛发展,肉制品鉴别有了真正敏感稳定而高效的检测手段,目前分子生物学检测方法主要包括常规PCR技术、多重PCR技术、限制性片段长度多态性PCR技术、DNA杂交与基因芯片技术、实时荧光PCR技术、数字PCR技术和DNA条形码技术,其中实时荧光PCR技术为现在监管部门对肉制品鉴别的常用技术手段。本文主要介绍这七种技术的研究与进展,对未来的研究方向进行了探讨和展望,为监管部门和科学研究者提供参考。     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.