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。     

A false single nucleotide polymorphism generated by gene duplication compromises meat traceability

Controlling meat traceability using SNPs is an effective method of ensuring food safety. We have analyzed several SNPs to create a panel for bovine genetic identification and traceability studies. One of these was the transversion g.329C>T (Genbank accession no. AJ496781) on the cytochrome P450 17A1 gene, which has been included in previously published panels. Using minisequencing reactions, we have tested 701 samples belonging to eight Spanish cattle breeds. Surprisingly, an excess of heterozygotes was detected, implying an extreme departure from Hardy-Weinberg equilibrium (P<0.001). By alignment analysis and sequencing, we detected that the g.329C>T SNP is a false positive polymorphism, which allows us to explain the inflated heterozygotic value. We recommend that this ambiguous SNP, as well as other polymorphisms located in this region, should not be used in identification, traceability or disease association studies. Annotation of these false SNPs should improve association studies and avoid misinterpretations.     

基于SNP标记的肉类溯源技术

本文阐述了建立肉类溯源系统对我国食品安全和国际贸易的重要性,并对溯源管理、食品溯源系统进行概述.重点讲述各种DNA标记技术的原理、利弊,以及在肉制品溯源标记应用的比较、SNP位点的检测方法等.最后指出了我国肉类溯源技术研究的方向和趋势.     

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。     

Effect of consanguinity on Argentinean Angus beef DNA traceability

Since the 1990s several authors have envisaged the use of DNA to certify meat origin. Two major parameters must be assessed before a DNA based traceability protocol can be implemented in the food chain: (i) the information content of a DNA marker set in a specific livestock breed or group of breeds; (ii) the minimum number of DNA markers needed to obtain a statistically acceptable match probability. The objective of the present work was to establish the effect of different levels of inbreeding in the matching efficiency, and the minimum number of microsatellite markers needed, in a DNA based meat traceability program, starting from an 11-microsatellite marker panel. Samples were obtained from beef production farms in South America, where animals are typically bred under pasture-based extensive conditions. Three groups of animals with different consanguinity rates were sampled. Exclusion power (Q) was higher than 0.999998 and match probability lower than 3.01E−08, for the whole set of markers within each group. Both values were affected by consanguinity. To reach a two mismatch criteria exclusion power (Q₂) of 99.99, six markers were needed in unrelated animals whereas seven markers were needed in related animals. To reach Q₂ = 99.9999, 8 and 10 microsatellite markers, respectively, were needed. In general, one or two more microsatellite markers were needed to identify consanguineous animals. This study proved the DNA marker set used to be suitable for the identification of the meat from all slaughtered animals in Argentina, per week, month, and year.     

Evaluation of microsatellites as a potential tool for product tracing of ground beef mixtures

Microsatellite genotyping was evaluated as a potential tool for DNA-based tracing of ground beef product. DNA from mixtures containing different numbers of individuals was analysed with a set of cattle microsatellite markers frequently used for parentage testing. As samples contained DNA from several animals, the microsatellite markers showed multiple peaks. The method could distinguish between mixtures containing equal amounts of meat from three different individuals, meat from three individuals mixed in different proportions, ground beef mixtures purchased in different cities, and different batches of ground beef patties. Limitations occurred when batches contained large numbers of individuals (>10) and different batches used meat from the same individuals. We conclude that DNA microsatellites may be useful for DNA traceability of ground beef mixtures prepared from less than 10 individuals, but where larger numbers of animals contribute to a mixture the method is not consistently accurate.     

Practical application of DNA fingerprinting to trace beef

DNA fingerprinting allows the verification of conventional methods used to implement beef traceability. At any point along the supply chain, the identity of an animal or piece of meat can be checked by comparison of its DNA profile with an initial sample. Practical application of DNA fingerprinting to trace beef requires a choice of DNA markers as well as the optimization of sampling methods. This has been achieved as the result of collaboration between meat technicians and geneticists over a period of 4 years. The discrimination power of nine highly polymorphic microsatellite markers was evaluated. We propose that three markers (with a 0.001 probability that two individual profiles match by chance) are adequate for routine tests. Two key points along the production-commercialization chain where sampling must be systematic were defined: (i) the tagging of the calf (identity control) and (ii) after slaughter (slaughter control), before the animal loses its external appearance. The identity control was blood collected on a filter paper adapted to the ear tag; the slaughter control was the tagged ear itself. These constituted the control samples, which were archived with a code matching the individual tag number. Test samples were obtained on a random basis from live animals, carcasses, and pieces of meat at cutting halls and at the retail outlet and in cases when the verification of identity was needed. The DNA profiles of the test samples and the controls were then obtained and compared, to verify either an individual identity or the origin of a piece of meat from the stated animal.     

Traceability of mussel (Mytilus chilensis) in southern Chile using microsatellite molecular markers and assignment algorithms. Exploratory survey

The international seafood trade has adopted the food chain or “from farm to fork” concept in terms of standards and regulations regarding food quality, safety and authenticity, from primary production to the consumer. This has led to an increasing need for traceability, but administrative traceability systems (physical labeling, information recording and automatic data treatment) are not flawless and require validation through analytical procedures. Currently, DNA-based methods used for species identification and population genetics, coupled with allocation algorithms can be used to verify administrative traceability systems. We evaluated the potential of a panel of nine microsatellite markers combined with allocation algorithms for their ability to assign Mytilus individuals from southern Chile to their geographical origin, evaluating the performance of four assignment methods: genetic distance and frequency-based criteria and a Bayesian based method using prior information or not. The reallocation test showed that the Bayesian method with prior information performed best. When tested with a real traceability verification case, the frequency-based algorithm showed the best results, re-allocating individuals to their original population at least 6 times more often than individuals from other locations in a challenging scenario with low genetic differentiation among locations. In order to apply this allocation method for traceability purposes, it would be necessary to strengthen this SSR panel with more informative loci and complement it with SNP markers.     

肉产品分子溯源标记的研究进展

食品溯源管理对食品生产、加工和消费过程起到重要的监管作用。肉产品作为饮食中主要的蛋白质来源,其安全问题受到高度重视。在众多的溯源技术方法中,本文介绍了DNA 溯源技术使用的分子标记的研究进展,对AFLP 标记、SSR 标记、SNP 标记3 类标记进行分析比较,并指出SNP 标记必将成为动物身份识别首选的分子标记。     

Vision-based method for tracking meat cuts in slaughterhouses

Meat traceability is important for linking process and quality parameters from the individual meat cuts back to the production data from the farmer that produced the animal. Current tracking systems rely on physical tagging, which is too intrusive for individual meat cuts in a slaughterhouse environment. In this article, we demonstrate a computer vision system for recognizing meat cuts at different points along a slaughterhouse production line. More specifically, we show that 211 pig loins can be identified correctly between two photo sessions. The pig loins undergo various perturbation scenarios (hanging, rough treatment and incorrect trimming) and our method is able to handle these perturbations gracefully. This study shows that the suggested vision-based approach to tracking is a promising alternative to the more intrusive methods currently available.     

Traceability of four European Protected Geographic Indication (PGI) beef products using Single Nucleotide Polymorphisms (SNP) and Bayesian statistics

The use of SNPs in combination with Bayesian statistics for the geographic traceability of cattle was evaluated using a dataset comprising 24 breeds from Italy, France, Spain, Denmark, the Netherlands, Switzerland and UK genotyped with 90 polymorphic markers.The percentage of correct assignment of the individuals to their Country of origin was 90%, with an average assignment probability of 93% and an average specificity of 92%. The higher value was observed for UK breeds (97% of correct assignment) while Swiss animals were the most difficult to allocate (77% of correct assignment).Tracing of Protected Geographic Indication (PGI) products, the approach correctly assigned 100% of Guaranteed Pure Highland Beef; 97% of “Vitellone dell’Appennino Centrale” breeds; 84% of Ternera de Navarra, and 80% of Boeuf de Chalosse.Methods to verify Products of Designated Origin (PDO) and Protected Geographic Indication (PGI) products will help to protect regional foods and promote the economic growth of marginal rural areas by encouraging the production of high quality niche market foods.     

Traceability of four European Protected Geographic Indication (PGI) beef products using Single Nucleotide Polymorphisms (SNP) and Bayesian statistics

The use of SNPs in combination with Bayesian statistics for the geographic traceability of cattle was evaluated using a dataset comprising 24 breeds from Italy, France, Spain, Denmark, the Netherlands, Switzerland and UK genotyped with 90 polymorphic markers.The percentage of correct assignment of the individuals to their Country of origin was 90%, with an average assignment probability of 93% and an average specificity of 92%. The higher value was observed for UK breeds (97% of correct assignment) while Swiss animals were the most difficult to allocate (77% of correct assignment).Tracing of Protected Geographic Indication (PGI) products, the approach correctly assigned 100% of Guaranteed Pure Highland Beef; 97% of “Vitellone dell’Appennino Centrale” breeds; 84% of Ternera de Navarra, and 80% of Boeuf de Chalosse.Methods to verify Products of Designated Origin (PDO) and Protected Geographic Indication (PGI) products will help to protect regional foods and promote the economic growth of marginal rural areas by encouraging the production of high quality niche market foods.     

Clostridium perfringens and its toxins in minced meat from Kars, Turkey

The aim of this study was to determine the prevalence of Clostridium perfringens and its toxins in minced meat. A total of 96 minced meat samples were collected from local markets (16) and small butcher's shops (80) in Kars (Turkey). Samples were analysed for the presence of C. perfringens and its toxins using a commercially available ELISA kit. A total of 31 (32%) Clostridium spp. strains were isolated and 17 (55%) of these isolates were identified as C. perfringens. Four (25%) of the samples from local markets and 27 (34%) from small butcher's shops were contaminated with Clostridium spp. Furthermore, C. perfringens was isolated from two (12%) and 15 (19%) samples from local markets and small butcher's shops, respectively. Mean counts of Clostridium spp. were 2.2 ± 0.83 × 10² CFU g^-1 for local markets and 4.35 ± 8.53 × 10² CFU g^-1 for small butcher's shops; mean counts for C. porringers were 2.75 ± 0.21 × 10² and 6.82 ± 10.96 × 10² CFU g^-1 from markets and butcher's shops, respectively. The number of samples contaminated with both Clostridium spp. and C. perfringens was higher in small butcher's shops than in local markets. Moreover, higher numbers of Clostridium spp. and C. perfringens were isolated in samples from small butcher's shops than from local markets. A total of 13 (13%) samples were also positive for toxins produced by the organism, as detected by ELISA. Eleven samples from small butcher's shops and two samples from local markets were positive for the C. perfringens toxins tested. Moreover, two (12%), one (1%), four (4%) and two (2%) samples were contaminated with C. perfringens types A, B, C and D, respectively. In conclusion, some meat samples collected from local markets and small butcher's shops contained C. perfringens and its toxins and, therefore, present a potential risk of food poisoning.     

High-resolution melting analysis: a promising molecular method for meat traceability

To find a promising molecular method for meat traceability, three methods of single nucleotide polymorphism (SNP) detection: RFLP-PCR analysis, high-resolution melting (HRM) analysis, and TaqMan probe analysis, have been compared in terms of accuracy, ease of use, throughput capability, and cost. We genotyped ten pork DNA samples across three SNPs. The results showed that the HRM genotyping method was the most accurate and easiest to use with the lowest cost, while TaqMan probe analysis provided similar results, but its cost was much higher.     

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.     

Effect-based detection of synthetic glucocorticoids in bovine urine

Challenges to testing for the illicit use of anabolic substances in meat-producing animals stem from the production of new synthetic compounds and the administration of low-dose cocktails to circumvent detection by the surveillance schemes of European Union member states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In order to verify the effect of natural corticosteroids on the method, the bioassay was tested first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose–response curves were measured for the most commonly used synthetic glucocorticoids. The bioassay’s ability to detect them in spiked and incurred samples of bovine urine was then evaluated. Finally, its performance was compared against a commercially available ELISA kit ordinarily used in screening activities. GR-CALUX performance did not appear to be influenced by physiological levels of endogenous corticosteroids in the farm samples, whereas an increase in these hormones might invalidate the analysis in samples obtained at the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ? dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the bioassay failed to detect the prohormone prednisone. The results obtained from analysis of the spiked and incurred specimens reproduced those of the blank samples and the pure compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in meat-producing bovines. Its ability to detect the most commonly used synthetic glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less susceptible to matrix effects than ELISA.     

Rapid evaporative ionisation mass spectrometry and chemometrics for high-throughput screening of growth promoters in meat producing animals

In a proof of concept perspective, Rapid Evaporative Ionisation Mass Spectrometry (REIMS) was explored for the direct analysis of meat samples from β-agonist treated livestock. In this context, the combination of REIMS with untargeted metabolomics was investigated to identify carcasses from treated animals on the basis of a modification of indirect metabolites profile. The REIMS analysis generated specific lipid profiles which enabled the differentiation of meat samples collected from pigs treated with ractopamine via their feeding regime. Furthermore, the strategy was found successful when tested on different muscle types (loin, shoulder and thigh), which further expands its applicability. Classification performances were greater than 95% accurate which fully answers requirements of a screening strategy. This research indicates that REIMS implemented in an untargeted-metabolomics workflow can be considered as a high-throughput and accurate strategy for real-time meat classification in relation to ractopamine (and wider β-agonists) treatment in pig production. This approach may subsequently be implemented as a rapid screening test, at the slaughterhouse or at border inspection points, to detect such practice.     

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.     

Pathways to increase consumer trust in meat as a safe and wholesome food

This paper focuses on the effect of information about meat safety and wholesomeness on consumer trust based on several studies with data collected in Belgium. The research is grounded in the observation that despite the abundant rise of information through labelling, traceability systems and quality assurance schemes, the effect on consumer trust in meat as a safe and wholesome product is only limited. The overload and complexity of information on food products results in misunderstanding and misinterpretation. Functional traceability attributes such as organisational efficiency and chain monitoring are considered to be highly important but not as a basis for market segmentation. However, process traceability attributes such as origin and production method are of interest for particular market segments as a response to meat quality concerns. Quality assurance schemes and associated labels have a poor impact on consumers' perception. It is argued that the high interest of retailers in such schemes is driven by procurement management efficiency rather than safety or overall quality. Future research could concentrate on the distribution of costs and benefits associated with meat quality initiatives among the chain participants.     

Marker imputation with low-density marker panels in Dutch Holstein cattle

The availability of high-density bovine genotyping arrays made implementation of genomic selection possible in dairy cattle. Development of low-density single nucleotide polymorphism (SNP) panels will allow the extension of genomic selection to a larger portion of the population. Prediction of ungenotyped markers, called imputation, is a strategy that allows using the same low-density chips for all traits (and for different breeds). In the present study, we evaluated the accuracy of imputation with low-density genotyping arrays in the Dutch Holstein population. Five different sizes of genotyping arrays were tested, from 384 to 6,000 SNP. According to marker density, the overall allelic imputation error rate obtained with the program DAGPHASE, which relies on linkage disequilibrium and linkage, ranged from 11.7 to 2.0%, and that obtained with the program CHROMIBD, which relies on linkage and the set of all genotyped ancestors, ranged from 10.7 to 3.3%. However, imputation efficiency was influenced by the relationship between low-density and high-density genotyped animals. Animals with both parents genotyped had particularly low imputation error rates: <1% with 1,500 SNP or more. In summary, missing marker alleles can be predicted with 3 to 4% errors with approximately 1 SNP/Mb (approximately 3,000 markers). The CHROMIBD program proved more efficient than DAGPHASE only at lower marker densities or when several genotyped ancestors were available. Future studies are required to measure the effect of these imputation error rates on accuracy of genomic selection with low-density SNP panels.