Genetic traceability of livestock products: A review

Traceability is the ability to maintain the identification of animal, or animal products, all along the production chain. It represents an essential tool to safeguard public and animal health and to valorize typical production systems. European food legislation is particularly strict and traceability systems, based on product labeling, have become mandatory in all European countries. However, the implementation of this system does not ensure consumers against fraud. Paper documents can be counterfeit so researchers have focused on the study of genetic traceability systems based on products identification through DNA analysis. In fact DNA is inalterable, detectable in every cell, resistant to heat treatments, and allows for individual, breed or species identification. Even if results are promising, these techniques are too expensive to be converted in routine tests but they could be a trusted tool for verification of suspected fraud. The present review proposes a synthesis of the major advances made in individual, breed, and species genetic identification in the last years, focusing on advantages and disadvantages and on their real future applications for animal productions.     

Application of microsatellite markers as potential tools for traceability of Girgentana goat breed dairy products

In livestock, breed assignment may play a key role in the certification of products linked to specific breeds. Traceability of farm animals and authentication of their products can contribute to improve breed profitability and sustainability of animal productions with significant impact on the rural economy of particular geographic areas and on breed and biodiversity conservation. With the goal of developing a breed genetic traceability system for Girgentana dairy products, the aim of this study was to identify specific microsatellite markers able to discriminate among the most important Sicilian dairy goat breeds, in order to detect possible adulteration in Girgentana dairy products. A total of 20 microsatellite markers were analyzed on 338 individual samples from Girgentana, Maltese, and Derivata di Siria goat breeds. Specific microsatellite markers useful for traceability of dairy products were identified. Eight microsatellite markers showed alleles present at the same time in Maltese and Derivata di Siria and absent in Girgentana and, therefore, they were tested on DNA pools of the three breeds. Considering the electropherograms' results, only FCB20, SRCRSP5, and TGLA122 markers were tested on DNA samples extracted from cheeses of Girgentana goat breed. These three microsatellite markers could be applied in a breed genetic traceability system of Girgentana dairy products in order to detect adulteration due to Maltese and Derivata di Siria goat breeds.     

Universal primers for species authentication of animal foodstuff in a single polymerase chain reaction

BACKGROUND: There are many DNA‐based systems for detecting animal species present in food and food products, applicable for food quality control and authentication. However, most (if not all) methods require more than one pair of primers and cannot be applied over a wide taxonomic range, e.g. identifying vertebrates and invertebrates with the same primers and protocols. RESULTS: A pair of primers is described here that allows in a single polymerase chain reaction the identification of animal species in food and processed (precooked, canned or smoked) food products over a wide taxonomic range. CONCLUSION: These primers permit the identification of most animal taxa employed in human nutrition, from invertebrates such as molluscs to higher vertebrates, distinguishing between species of the same genus. The short fragment amplified within the 16S rDNA exhibits phylogenetic value and could be considered universal based on the wide taxonomic range assayed. The primers are easy to use and accessible for laboratories with a modest budget, as well as being valuable for consumer information and to reveal food fraud. Copyright © 2012 Society of Chemical Industry     

SNP标记用于猪肉产品DNA溯源

采用基于个体基因组DNA序列的差异而进行个体识别的DNA溯源技术,建立DNA溯源系统对肉产品的质量安全进行控制。在实验群体中(10个品种,233个个体)检测了33个新单核苷酸多态性(single nucleotide polymorphism,SNP)标记的遗传多样性,通过杂合度计算筛选出6个SNP标记可用于猪肉产品DNA溯源。进一步在屠宰场采样进行溯源模拟实验,结果表明筛选的18个SNP标记(6个新SNP标记结合已有的12个SNP标记)能有效区分100头猪个体,随机抽取的10个个体的组织样品都能通过基因型比对找到对应的个体。本研究可为早日建立猪肉产品的DNA溯源系统提供一定的技术参考。     

Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR

Food and feed traceability has become a priority for governments due to consumer demand for comprehensive and integrated safety policies. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for specific detection of rabbit and hare material in animal feeds and pet foods. The technique is based on the use of three species-specific primer/probe detection systems targeting three 12S rRNA gene fragments: one from rabbit species, another one from hare species and a third fragment common to rabbit and hare (62, 102 and 75 bp length, respectively). A nuclear 18S rRNA PCR system, detecting a 77-bp amplicon, was used as positive amplification control. Assay performance and sensitivity were assessed through the analysis of a batch of laboratory-scale feeds treated at 133°C at 3 bar for 20 min to reproduce feed processing conditions dictated by European regulations. Successful detection of highly degraded rabbit and hare material was achieved at the lowest target concentration assayed (0.1%). Furthermore, the method was applied to 96 processed commercial pet food products to determine whether correct labelling had been used at the market level. The reported real-time PCR technique detected the presence of rabbit tissues in 80 of the 96 samples analysed (83.3%), indicating a possible labelling fraud in some pet foods. The real-time PCR method reported may be a useful tool for traceability purposes within the framework of feed control.     

Post-slaughter traceability

Traceability programs can cover the whole of life, or parts of it, for individual animals or groups/lots of animals. Of 13 country or community traceability programs for cattle/beef, 11 are mandatory (4 encompass, or are scheduled to encompass, birth to retail; 7 cover birth to slaughter) while 2 are voluntary and encompass birth to slaughter. Of 10 country or community traceability programs for swine/pork, 2 are mandatory (1 covers birth to retail; 1 covers birth to slaughter) while 8 are voluntary. Of 6 country or community traceability programs for sheep/sheep-meat, 3 are mandatory (1 encompasses birth to retail; 2 encompass birth to slaughter) while 3 are voluntary. Mandatory birth to retail programs that include "post-slaughter individual animal identification (IAID) traceability" have been implemented for cattle/beef, swine/pork and sheep/sheep-meat by the European Union and for cattle/beef by Japan. Many of the voluntary as well as mandatory, birth to slaughter traceability programs for all three species are presumed (though that is not specified) to include "post-slaughter group/lot identification (GLID) traceability" - e.g., those qualifying products for shipment to the European Union. "Post-slaughter IAID traceability" can be accomplished in very-small, small, medium, large and very-large packing plants using single-carcass processing units, tagging and separation/segregation, and/or deoxyribonucleic acid (DNA) fingerprinting technology but all of these approaches are time-consuming and costly; and, to-date, in most countries, there has been no reason compelling enough to cause industry to adopt such protocols or technology.     

AN EFFICIENT DNA-EXTRACTION PROTOCOL FOR NUT SEEDS

Establishing genetic origin of food products allows verification of the authenticity of valuable foods and discourages adulteration with material of lower cost and value. This is particularly important for food products that have obtained European recognition. The use of molecular markers could be a solution for species and cultivar identification and for the genetic traceability. The development of efficient DNA-extraction protocols is an essential step for the procedure. In this work, a method for total DNA isolation was developed for hazelnut, almond and walnut seeds. The efficiency and reliability of the method was tested by assessing quantity and quality of the extracted DNA, and by Polymerase Chain Reaction (PCR) amplification, using two decamer primers and three universal primer pairs designed on the chloroplast DNA. The success of amplifications confirmed the presence of both nuclear and chloroplast DNA in the extracted sample.

PRACTICAL APPLICATIONS

Cultivar identification of nuts, based on morphological traits only, is often difficult, and adulterations with seeds of lower cost and quality are easy, above all when they are sold as shelled kernel, as is common for hazelnut. The genetic identification of cultivars is nowadays a routine practice, because of the development of DNA-typing techniques based on molecular markers. An efficient DNA-extraction procedure for hazelnut, almond and walnut seeds is a preliminary step required for enabling the recognition of the cultivar of origin of the nuts and fighting commercial frauds. It will also be useful in marker-assisted selection, applied in breeding.     

DNA条形码技术在肉及其制品中的应用

DNA条形码技术作为一种新的分子生物学检测技术在鉴定和区分各物种和物种间亲缘关系方面得到了广泛应用,该技术能实现对肉的快速、准确检测。DNA条形码已成为生物学领域发展最迅速的一种技术,该技术基于广泛的物种基因数据库信息,在肉品研究中具有良好的应用前景。本文简述DNA条形码技术的基本原理,并基于DNA水平上与其他相关技术进行比较,综述其在物种鉴定、肉品质量安全、商业欺诈等方面的应用,并对该技术在今后肉品科学研究中的应用进行展望。      

Species authentication of octopus,cuttlefish, bobtail and bottle squids (families Octopodidae,Sepiidae and Sepiolidae) by FINS methodology in seafoods

In the present work a molecular a method for the authentication of cephalopods products was developed, which allows the genetic identification of about 30 species belonging to the families Octopodidae, Sepiidae and Sepiolidae. This molecular system is based on the phylogenetic analysis of DNA sequences. The molecular marker studied was the cytochrome b gene (cyt b), that was amplified by PCR and subsequently sequenced. The developed methodology was validated and further applied to 20 commercial samples, detecting 6 that were incorrectly labelled (30%). Therefore, this molecular tool could be applied in questions related to correct labelling, traceability, and import control of products containing the taxonomic groups studied.     

Meat traceability using DNA markers: application to the beef industry

Consumer concerns about beef demands instruments to assure its traceability. A methodology using DNA markers is proposed for beef identification focussing on a Spanish beef certification, Ternera de Navarra (Beef of Navarra). To validate this methodology the number of markers used and the implications of population structure in individual identification were evaluated. In order to get practical implementation, the sampling levels required, depending on the number of markers and amount of possible fraud, is also discussed. Using at least eight very informative markers the origin of retailed meat is always found independent of genetic population structure. The total control of fraud would be very expensive using large-scale application of DNA analyses and a strategy based on anonymous sampling is proposed.     

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。     

柑橘属产品真实性溯源技术研究现状及展望

近年来在柑橘的生产销售整个链条上，柑橘品种和产地的混淆造假以及以次充好等质量问题突出，所以研究柑橘属产品真实性溯源技术很有现实意义，目前已有的报道尚缺乏对柑橘属产品真实性溯源技术研究进展的系统性梳理和总结。该研究重点梳理和综述了稳定同位素和多元素分析、代谢组学分析、风味物质分析、光谱分析及核酸扩增检测等技术在柑橘属产品真实性溯源研究中的应用，并总结了各类溯源技术的优缺点，提出了目前有关湖北省脐橙的产地和品质等级鉴别研究存在的不足，同时对今后的研究方向作出了展望。以期为湖北省脐橙的产地溯源和质量监管提供一定的技术支撑。     

DNA barcoding detects market substitution in North American seafood

Seafood authentication and food safety concerns are a growing issue in today’s global marketplace, although traditional morphology-based identification keys and existing molecular approaches have limitations for species identification. Recently, DNA barcoding has gained support as a rapid, cost-effective and broadly applicable molecular diagnostic technique for this purpose. However, the maturity of the barcode database as a tool for seafood authentication has yet to be tested using real market samples. The present case study was undertaken for this reason. Though the database is undergoing continual development, it was able to provide species matches of >97% sequence similarity for 90 of 91 samples tested. Twenty-five percent of the samples were potentially mislabeled, demonstrating that DNA barcodes are already a powerful tool for the identification of seafood to the species level. We conclude that barcodes have broad applicability for authenticity testing and the phylogeographic patterning of genetic diversity can also inform aspects of traceability.     

SSR在猪个体识别及肉产品溯源中的应用

DNA溯源技术是根据动物个体之间遗传物质DNA序列的差异而进行个体识别并追溯到原产地的一种溯源技术。在试验群体中(11个品种,192个体)检测了24个SSR标记的遗传多样性,通过杂合度和多态信息含量计算筛选出11个SSR标记可用于猪肉产品的DNA溯源。在此基础上进一步在屠宰场采样进行了溯源模拟实验,结果表明筛选的11个SSR标记能区分100个个体,10份组织样品的SSR标记基因型是一一对应的,并且和43号个体基因型匹配。研究表明SSR标记可以用于猪个体识别和猪肉产品的溯源。     

Traceability from a European perspective

At pan-European level there is a need for traceability systems giving information on origin, processing, retailing and final destination of foodstuffs. Such systems shall enhance consumer confidence in food; enable the regulatory authorities to identify and to withdraw health hazardous and non-consumable foodstuffs from the market. Animal feeds are an element in this "food-to-farm" approach to public health. Such feedstuffs are preliminary elements of some foods for human consumption, and hence are an inherent element of the food chain. A harmonised pan-European food traceability protocol would greatly assist authorities in detecting fraud as well as dangerous substances. The food chain comprises a range of sequential and parallel stages bridging the full spectrum from agricultural production to the consumable foodstuffs by consumers. EU legislation on traceability and the technologies needed to implement this system for meat and meat products are the focus of this paper.     

Genetic approaches and technologies for improving the sustainability of livestock production

Livestock production industries worldwide face considerable conflicting challenges and pressures. In developed countries the challenge is to remain sustainable and competitive in the face of declining prices and increasing costs, competition and public pressures. In developing countries the strong increase in demand for livestock products must be met in circumstances where infrastructure is often minimal, there are limitations on inputs and the environment places demands on management and on the adaptive fitness of the livestock. In both situations, solutions to these problems must be sustainable and appropriate, yet be technically feasible, cost‐effective and publicly acceptable. This paper summarises the impact of two technologies that will make considerable contributions to sustainable livestock production systems, namely information technology and genetic technologies that utilise naturally occurring genetic variation. Genetic technologies are inherently sustainable owing to the permanent and cumulative nature of genetic change, and range from simple to sophisticated. They include breed choice, within‐breed selection and the use of genetic markers linked to gene variants conferring favourable attributes. Breeding goals include increased output, where required, enhanced product quality and increased disease resistance. These goals are illustrated by examples for the hill sheep and pig sectors in the UK and by challenges facing animal health in developing countries. Central to all examples is the gathering, management and interpretation of information, ie information technology, which enables rational genetic and management decisions to be made. Additionally, in all sustainable livestock production systems the maintenance and utilisation of biodiversity will help manage the risks of today as well as the challenges of the future. Copyright © 2004 Society of Chemical Industry     

A melanocortin 1 receptor (MC1R) gene polymorphism is useful for authentication of Massese sheep dairy products

Massese is an Italian sheep breed, with black or grey coat colour, mainly reared in the Tuscany and Emilia Romagna regions. Recently, the emerging interests in this breed have resulted in the production of Pecorino cheese obtained with only Massese milk. In order to be profitable, this marketing link between Massese breed and its products should be defended against fraudsters who could include milk of other sheep breeds or cow milk in Massese labelled productions. To identify the genetic factors affecting coat colour in sheep, we have recently analysed the melanocortin 1 receptor (MC1R) gene and identified several single nucleotide polymorphisms (SNPs). In this work, as a first step to set up a DNA based protocol for authentication of Massese dairy products, we further investigated the presence and distribution of one of these SNPs (c.-31G>A) in 143 Massese sheep and in another 13 sheep breeds (for a total of 351 animals). The Massese breed was fixed for allele c.-31A, whereas in all other breeds allele c.-31 G was the most frequent or with frequency of 0?·50. At the same nucleotide position the cattle MC1R gene carries the G nucleotide. Using these data we developed a method to detect adulterating milk (from other sheep breeds or from cow) in Massese dairy products based on the analysis of the c.-31G>A SNP. We first tested the sensitivity of the protocol and then applied it to analyse DNA extracted from ricotta and Pecorino cheese obtained with only Massese milk or obtained with unrestricted sheep and cattle milk. To our knowledge, this system represents the first one that can be used for breed authentication of a sheep production and that, at the same time, can reveal frauds derived from the admixture of milk of an unreported species.     

Quantitative competitive (QC) PCR for quantification of porcine DNA

Many meat products nowadays may contain several species in different proportions. To protect consumers from fraud and misdeclarations, not only a qualitative but also a quantitative monitoring of ingredients of complex food products is necessary. DNA based techniques like the polymerase chain reaction (PCR) are widely used for identification of species but no answer to the proportional amount of a certain species could be given using current techniques. In this study we report the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of porcine DNA using a new porcine specific PCR system based on the growth hormone gene of sus scrofa. A DNA competitor differing by 30 bp in length from the porcine target sequence was constructed and used for PCR together with the target DNA. Specificity of the new primers was evaluated with DNA from cattle, sheep, chicken and turkey. The competitor concentration was adjusted to porcine DNA contents of 2 or 20% by coamplification of mixtures containing porcine and corresponding amounts of bovine DNA in defined ratios.     

Genetic control of conventional labeling through the bovine meat production chain by single nucleotide polymorphisms using real-time PCR

Since January 2002, the European Union has adopted precise guidelines aimed at protecting the safety of meat and controlling the production chain. To this purpose, the conventional traceability of livestock and meat represents the main tool, but verification of traceability requires genetic support. At present, single nucleotide polymorphisms (SNPs) represent the most innovative molecular markers in genotyping studies. The aim of this study was to verify correct labeling in a bovine meat production chain by a real-time PCR protocol based on SNP analysis. Reference hair samples from 5,000 animals were randomly collected from 22 farms. Twelve hundred meat samples were collected at different steps of the bovine meat production chain. In particular, 1,000 meat samples were collected at the slaughterhouse and 200 samples from the same animals directly at the butcher's shop. The protocol was optimized and validated by testing a set of 16 SNP markers on 95 DNA samples from bovine sires of different breeds. Thereafter, the genotyping of 2,200 samples was conducted with a set of 12 selected SNPs to verify traceability of the meat production chain at three different stages: farm, slaughterhouse, and butcher's shop. Irregularities in conventional traceability were evidenced directly in 1.87% of the samples at the slaughterhouse. This percentage increased to 3.25% when sampling was conducted at the butcher's shop. This study demonstrates that despite the precautions adopted over the meat production chain, some critical points still exist that cause the loss of a correct association between registration numbers and samples.     

Short communication: Detection of undeclared presence of bovine milk in buffalo yogurt

The authenticity of buffalo (Bubalus bubalis) dairy products is a focal issue, considering the increasing demand for buffalo milk products. Therefore, the aim of this study was to investigate the undeclared presence of bovine (Bos taurus) milk in buffalo yogurt, to understand which risk factors might make the product vulnerable to fraud. Real-time PCR assay showed the undeclared presence of bovine DNA in addition to buffalo DNA in 18 of 72 samples. Given the widespread lack of data on the presence of undeclared milk species in buffalo dairy products, the study provides a significant insight into the incidence of fraud in the buffalo dairy field. The data from this study could help improve the analysis of food safety risks along the buffalo milk supply chain and in the dairy processing industry, perceived as being highly vulnerable to food fraud, and prioritize target areas for food policy making to steer and enforce European food fraud regulations.