Article

A DNA macro-array to simultaneously identify 32 meat species in food samples

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Considering the recent cases of meat adulteration and fraud, efficient and accurate analytical methods are needed for the specific identification of meat species as a key importance to maintain consumers' trust and to comply with various local labelling legislations. As a reference approach, the application of PCR to meat speciation is always limited to few species, whereas there is a strong need to broaden the number of identified meat species. PCR was here associated with a commercial DNA macro-array spotted with meat-specific capture probes, allowing the simultaneous identification of 32 meat species on 8 samples per chip support. Tested on pure meat samples, spiked samples, proficiency test samples, and processed samples, the method showed high specificity on the targeted species and allowed a sensitivity down to 1% (w/w). Easy-to-use, fast and cost-efficient, this multi-screening approach is fit-for-purpose as a tool to detect meat substitution or adulteration for meat testing laboratories.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Moreover, the direct PCR method is still time-consuming, requires complex operation and gel electrophoresis, and cannot be accurately quantified (Li, Jalbani et al., 2019;Shabani et al., 2015) through spectrophotometric methods because the PCR prod-ucts easily undergo interference with single-stranded DNA, RNA, proteins, and so on (Kang & Tanaka, 2018), which restricts its application in industry and commercial settings . Therefore, an upgraded technology has been developed in recent years, that is PCR associated with biochips (Cottenet, Sonnard, Blancpain, Ho, Leong, & Chuah, 2016;Iwobi, Huber, Hauner, Miller, & Busch, 2011;. The DNA biochip technologies have the advantages of the high sensitive and simultaneous detection of multispecies compared to simplex PCR methods (Cottenet et al., 2016;Iwobi et al., 2011). ...
... Therefore, an upgraded technology has been developed in recent years, that is PCR associated with biochips (Cottenet, Sonnard, Blancpain, Ho, Leong, & Chuah, 2016;Iwobi, Huber, Hauner, Miller, & Busch, 2011;. The DNA biochip technologies have the advantages of the high sensitive and simultaneous detection of multispecies compared to simplex PCR methods (Cottenet et al., 2016;Iwobi et al., 2011). Recently, developed two independent multiplex PCR methods based on 12S rRNA, 16S rRNA, ND2, and COI. ...
Article
Meat adulteration, mainly for the purpose of economic pursuit, is widespread and leads to serious public health risks, religious violations, and moral loss. Rapid, effective, accurate, and reliable detection technologies are keys to effectively supervising meat adulteration. Considering the importance and rapid advances in meat adulteration detection technologies, a comprehensive review to summarize the recent progress in this area and to suggest directions for future progress is beneficial. In this review, destructive meat adulteration technologies based on DNA, protein, and metabolite analyses and nondestructive technologies based on spectroscopy were comparatively analyzed. The advantages and disadvantages, application situations of these technologies were discussed. In the future, determining suitable indicators or markers is particularly important for destructive methods. To improve sensitivity and save time, new interdisciplinary technologies, such as biochips and biosensors, are promising for application in the future. For nondestructive techniques, convenient and effective chemometric models are crucial, and the development of portable devices based on these technologies for onsite monitoring is a future trend. Moreover, omics technologies, especially proteomics, are important methods in laboratory detection because they enable multispecies detection and unknown target screening by using mass spectrometry databases.
... 28 Furthermore, a species-specic PCR method was reported to identify pork, horse meat, and donkey meat, achieving a limit of detection (LOD) as low as 0.1% w/w donkey meat in beef. 29 Besides, real-time PCR, 23 multiplex PCR, 30,31 PCR-DNA pyrosequencing, 32 DNA microarray 33 and others have also been reported for species-specic detection of donkey meat. Nevertheless, their methods bear some limitations, such as a limited number of species for specicity test, 29 relatively labour intensive bench work (e.g., electrophoresis), 28 and the use of expensive equipment. ...
... Thus, the results from all prediction and experiments demonstrate the high specicity of the designed primer against the common meats (beef, pork, etc.) and vegetables (carrot, green pepper, etc.) as well as some meats of uncommon animal found in previous records as adulterants (fox, mouse, cat, etc.) involved in the meat authentic issue related to donkey meat. Compared to the scope of the previous studies in which mainly focused on major meats available in common grocery stores in developed countries, 6,7,23,27,28,31,33 our current study also included many species that are poorly monitored but do exist in meat products produced in Africa 9 and Asia (internal data). This provides a more powerful tool for regulatory agencies with high condence in reducing the false positive issue during the routine tests. ...
Article
Full-text available
Food authenticity is a global issue and has raised increasing concerns in the past decades. DNA-based methods are more favourable than the conventional protein-based techniques and have been applied to species identification and meat fraud detection. To effectively identify donkey meat for meat product authentication, a highly specific and robust method that coupled polymerase chain reaction (PCR) with lateral flow immunoassay (LFI) was developed. Donkey-specific PCR primers were designed by targeting at the mitochondrial D-loop gene and the specificity was verified in silico and in vitro against 22 species involved in meat authentication. A limit of detection of 0.0013 ng μL-1 DNA extract was achieved and as low as 0.001% w/w (raw) and 0.01% w/w (cooked) donkey meat in beef were successfully detected using the developed PCR-LFI. LFI strip-based visualization of PCR products allowed for a 10-fold higher sensitivity than conventional gel electrophoresis and significantly reduced the analysis time for the post-PCR analysis. This PCR-LFI is highly suitable for rapid identification of donkey or incorporating into multiplex screening protocol for other meat authentication in the laboratories of both regulatory agencies and commercial services.
... Further, chemical profiles may vary along with the changes in the environment, harvest time, storage condition, and processing methods (Woolfe & Primrose, 2004), making the entire analysis to be more complicated and technical challenging. DNA-based methods have received increasing attention for the authenticity test of meat products due to high sensitivity and specificity (Lo & Shaw, 2018), including species-specific polymerase chain reaction (PCR) (Amaral et al., 2015;Kesmen, Yetiman, Şahin, & Yetim, 2012), DNA hybridization (Cottenet et al., 2016), and sequencing (Bertolini et al., 2015). methods, DNA is present in the majority of cells and identical information can be obtained regardless of the physiological condition or tissue type of the sample (Lo & Shaw, 2018). ...
Article
A simple PCR-based lateral flow assay (LFA) was developed to detect turkey ingredient in foods. We developed this assay based on PCR amplification of turkey DNA, followed by detecting labeled amplicon by LFA. The results could be observed by the naked eye within 5 min following PCR. The reliability of this assay was confirmed by gel electrophoresis. Specificity tests validated no cross-reactivity with 21 other animal and plant samples. As low as 0.1% (w/w) of turkey in mixed meats were detected by the proposed PCR-LFA. Heat treatment did not affect the sensitivity of this assay for detecting turkey ingredient. The reproducibility of PCR-LFA was confirmed by the fully consistent intra-laboratory tests and acceptable inter-laboratory tests. Finally, this assay was successfully used to authenticate 24 commercial turkey products. No turkey ingredient was identified in two frozen turkey rolls, indicating food fraud cases. PCR-LFA provides an accurate, sensitive and cost-effective approach for turkey authentication. It is a promising tool for species authentication, food quality assurance and traceability.
... The importance of preparing a highly sensitive, inexpensive and rapid method led to design the animal species-specific detection system, which is able to detect simultaneously more animal species which is normally used in multiplex PCR. Cottenet et al. [30] described a macro-LCD array system for detection of different meat species in food and its evaluation needed LCD array slide scanner and corresponding software. The disadvantage of this method compared to the presented method is that it needs special equipment which cannot normally be found in all laboratories worldwide. ...
Article
Full-text available
Animal products play a significant role in human consumption. Therefore, identifying the food fraud is of great importance. We describe a simple but an innovative method for the simultaneous detection of meat sources from different animal species (cattle, buffalo, sheep, goat, horse, camel, pig, dog, cat, rabbit, chicken, wild duck and turkey) and human as a control DNA was extracted from samples prepared from human and above-mentioned animals and amplified using a common primer pair derived from a region of the mitochondrial DNA. The PCR products were subsequently hybridized with the species-specific DNA probes covalently bonded to a Biodyne C blotting membrane. The results showed that the PCR products generated from mammalian reacted as expected, to catch-all probes, mammalian catch-all probes and the corresponding species specific DNA probes but not with bird catch-all probes. The avian PCR product reacted also as expected, to catch-all probes, bird catch-all probes and the corresponding species-specific DNA probes, and not with the mammalian catch-all probes. These results showed that the used detection system could discriminate simultaneously, each animal species from the others. To determine the sensitivity of the presented hybridization method, the meat samples from 2 to 6 animals were mixed and the extracted DNA was analyzed. Interestingly, the species-specific reaction could be detected in all mixtures consisting of different species. Furthermore, it could be shown that the mixed meat DNA ratio 1/100 also could be detected simultaneously.
... Cabe destacar la sustitución fraudulenta de las carnes, por ejemplo carne de burro adulterada con carne de zorro, de cordero con rata y cabra, de res con jirafa, canguro y caballo, de pollo o conejo con gato, de venado con cordero, de cabra con perro y gato 341,342 . ...
Book
Full-text available
La alimentación humana está condicionada por factores ideológicos, climáticos, geográficos, tecnológicos y religiosos, entre otros. Esos factores crean patrones de dieta que los humanos eligen por variedad de razones, como preocupaciones éticas, deseo de una mejor salud, creencias religiosas y consideraciones ambientales, entre otras. El condicionamiento en la alimentación es creado en un ambiente sociocultural, que dista en tiempo, de la alimentación originaria en un ambiente natural; la cual por modificaciones conductuales, en la especie humana, evolutivamente, han causado cambios en adaptaciones anatómicas y fisiológicas en humanos. El estudio de esas adaptaciones genera hipótesis enmarcadas dentro de un contexto con validez bajo distintos puntos de vista, lo que dificulta establecer, en parte, la verdad sobre la alimentación humana. Para la elaboración de este libro se consultó literatura puramente científica. Los temas tratados abarcan la adaptación evolutiva de primates humanos, no humanos y algunas otras especies, existentes y extintas, con énfasis en la anatomía comparada; la experiencia multisensorial en la comunicación entre plantas y animales; la evolución de la dieta humana y aspectos nutricionales y de salud asociados al procesamiento de alimentos, al consumo de macronutrientes de origen animal y vegetal, y de compuestos fito y zooquímicos; además, relaciones entre la microbiota intestinal y las dietas. Temática que ofrece al lector diversidad de argumentos que llevan a suponer sobre lo que los humanos modernos, deberían o no, comer.
... DPCR works by partitioning a sample of nucleic acids into many individual, parallel PCR reactions. This separation permits a more reliable and sensitive [45] Real time PCR Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration [93] Real time PCR Identification of meat species in pet foods using a real-time polymerase chain reaction (PCR) assay [59] Real-time PCR Identification of species in ground meat products sold on the U.S. commercial market using DNAbased methods [94] Species-specific PCR Establishment and application of a polymerase chain reaction for the identification of beef [63] Species-specific PCR Identification of goose, mule duck, turkey, and swine in foie grass by species-specific polymerase chain reaction [61] Species-specific PCR PCR assay for the identification of animal species in cooked sausages [62] Species-specific PCR Authentication of a traditional game meat sausage (Alheira) by species-specific PCR assays to detect hare, rabbit, red deer, pork and cow meats [64] Species-specific PCR Identification of duck, partridge, pheasant, quail, chicken and turkey meats by species-specific PCR assays to assess the authenticity of traditional game meat Alheira sausages [95] Species-specific PCR Applicability of species-specific polymerase chain reaction for fraud identification in raw ground meat commercially sold in Iran [96] DNA sequencing Identification of sika deer and red deer using partial cytochrome b and12S ribosomal RNA genes [97] DNA sequencing A high incidence of species substitution and mis-labelling detected in meat products sold in South Africa [65] DNA macro-array A DNA macro-array to simultaneously identify 32 meat species in food samples [98] droplet digital PCR (ddPCR) Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR) [43] Digital PCR An integrated microfluidic system for bovine DNA purification and digital PCR detection [71] Fig. 2 Routine method identification for species meat products 1 3 measurement of nucleic acid amounts. DPCR is also more tolerant of inhibitors than real time PCR [68]. ...
Article
Full-text available
Meat species identification and animal authentication in meat products is a significant subject, attention to which would contribute to fair-trade, and would enable consumers to make informed choices. Analytical methods are often based on protein or DNA measurements. Methods based on protein fractions include electrophoretic, chromatographic and immunological techniques and are often not suitable for compound food products, nor are they sensitive in processed products to differentiate closely related meat species. Advances in DNA technology have led to the rapid development of alternative approaches to species identification. Recently, application of polymerase chain reaction in food analysis has increased in the light of their simplicity, specificity and sensitivity. This review discusses a wide range of analytical methods with a focus on their ability to quantify meat and authentication of meat products.
... DNA array is a molecular technique which enables simultaneous detection of multiple targets based on hybridization of the probe and the sample DNA. For example, 32 meat species can be analyzed using DNA macroarray (Cottenet et al., 2016), commercially available fish products (Kochzius et al., 2008), meat products (Peter, Brünen-Nieweler, Cammann, & Börchers, 2004) and various degraded food samples (Teletchea, Bernillon, Duffraisse, Laudet, & Hänni, 2008) can be identified by DNA microarray. The greater number of probes on the chips in DNA microarray provides higher throughput in detection compared with smaller number of probes in DNA macroarray. ...
Article
Contamination of vegan products and vegan shrimps with real shrimps and crustaceans must be avoided to comply with vegan claims, even more for preventing strong allergenic reactions. Therefore, the detection of crustaceans must be reliable and sensitive enough for authenticity, traceability, and food safety purposes. A new real-time PCR assay was developed targeting the mitochondrial 16S rRNA gene of crustaceans and was optimised to avoid critical mismatches with primers and probe. By testing several crustacean species and common food ingredients, the method was demonstrated to be specific to crustaceans only. To comply with the limit of non-vegan contamination established at 0.1% (w/w) by the European Vegetarian Union, dedicated cut-off CT values were determined on vegan raw materials and on vegan shrimps spiked with crustacean materials. The method reached a sensitivity ≤ 0.0005% (w/w), which was further confirmed on reference materials containing a similar amount of crustacean.
Chapter
A current problem in the food and agricultural industry is the detection of animal species substitution. Emphasis is placed on the selection of simple, reliable, reproducible and repeatable methods for identifying and quantifying food ingredients. For this reason, it is necessary to know about effective systems to ensure effective control, correct identification, and quantification of meat and its possible replacement. This chapter focuses on the design of a system for identifying and quantifying the DNA of the animal species from which the meat originated and possible meat substitutes. Thermal treatment of the products and their mechanical processing was also considered in order to obtain relevant and repeatable results for several samples simultaneously so that several aspects are examined in one analysis at a time. The Meat 5.0 LCD-Array kit was verified in this chapter first. To eliminate cross-reaction and to evaluate the specificity of the method, pure samples were tested for each animal species included in the analysis. Tissue samples from 21 species of animals, including domestic livestock, wildlife and avian species, were used as biological material. Specific analyses were focussed on pig meat. Furthermore, 11 blends of a combination of three animal species were analyzed to verify functionality, adding two adulterants to the main blend constituent of pork in three different ratios. The animal species under investigation were identified by their specific probes with 100% specificity. Tests were also performed on samples after various heat treatments, with no observed changes, which means that the kit could also be used to test cooked or sterilized foods. It was not possible to quantify the DNA using the Meat 5.0 LCD-Array. Also, the LCD-Array kit has been evaluated as successful in identifying non-meat animal proteins. The innuDETECT Assay was verified as a second. The combination of 6 binary mixtures of meats in eighteen different concentration orders of the addition was analyzed. The addition concentration range was from 100 to 0.1%. All analyzed animal species included in the experiment were equally correctly identified after reaching the 5% addition limit. In the first binary mixture where pork was added to the beef, the pig DNA was detected at 0.5% of the addition. In the second mixture, the chicken was identified only to a level of 5% of the addition of chicken to pork. In the third binary mixture, the turkey DNA was detected to a level of 1% of the addition of turkey to pork. In the fourth binary mixture where sheep was added to pork, the sheep DNA was captured to a level of 1% addition. In the fifth combination of goat and pork meat, goat DNA was captured at 1% of the addition. In the sixth binary mixture combination of beef and pork was able to capture the bovine DNA to a level of 0.5% addition. The results were identical for both studied mixtures before and after the heat treatment. This means that the heat treatment has no effect on the performance of the detection kits. These validated kits for the identification of animal species in food have a high potential in the field of laboratory food analysis and food control. These compared techniques are highly specific and allow easy identification of animal species, sufficiently sensitive and provide repeatable results.
Article
Consumption of plant-based products and meat alternatives has been rising for a few years, with vegetarian and vegan diets gaining more and more popularity. To comply with legislation and maintain consumers’ trust, food manufacturers of vegan products must ensure the absence of animal residues in these products. A real-time PCR method targeting the 16S mitochondrial rRNA gene from vertebrates has been developed in order to detect the presence of vertebrate material in plant-based ingredients and finished products. The reaction successfully amplified on mammals, birds, reptiles and fish samples only. The analysis of plant-based samples spiked at 0.1% (w/w) with meat or fish materials confirmed the sensitivity of the method at this level, considered as the limit for unintended presence of animal residues by the European Vegetarian Union. The method was applied to commercialized vegan finished products from European markets and confirmed the absence of animal material in all the tested samples.
Article
A molecular approach (DNA barcoding and phylogenetic analysis) using mitochondrial COI and 16SrRNA genes was used to identify species in crocodile dried food products (meat and feet) and skin sold on the Chinese market and generically labelled as “鳄鱼” (crocodiles). All the 80 collected samples (100%) were identified at species level and five of them were also identified at sub-species level using the COI gene. Limits of the DNA barcoding approach related to the presence of sequences from misidentified specimens on official genetic databases (Genbank and BOLD) were encountered. The only DNA barcoding method was successfully applied for the species identification of 47 (58.7%) samples (42 using the COI and 5 using the 16SrRNA) while the support of the phylogenetic analysis was considered in 7 (8.7%) samples (performed using the 16SrRNA gene). For the remaining 26 samples (43.3%) the species identification was only achieved by phylogenetic analysis using the COI gene. Three species were overall detected: Crocodiles siamensis (n= 44; 55%), C. porosus (n= 29; 36.2%) and Caiman crocodilus (n= 7; 8.7%) with the sub-species C. crocodylus crocodylus in 5 out of the 7 cases. Although the traceability system of these products in China presented evident shortcomings, outcomes from this study appeared comforting since all the three species are among the most reared for meat production and can plausibly feed the market requests. Interestingly, only one of these species is included among those considered by the new EU legislation on reptile meat. Therefore, although Chinese crocodilian-based products are still not allowed to be imported in the EU market, a future law up-dating could not be excluded considering the relevance of the Chinese exports for the EU. Outcomes from this study, other than allowing to monitor products through the whole food value chain, contribute to enrich the scientific pool of data from which EU food imports legislation draw upon.
Article
Adulteration of high-quality meat with their cheaper counterparts can be minimized by rapid and reliable methods for detecting meat species. Here an isothermal cross-primer amplification (CPA) technique combined with colloidal gold nucleic acid test strips (CPA strips) was developed to differentiate cow, sheep, arctic fox, and pig meat. A simple primer design for multiplex differentiation using a universal single-labeled CPA primer system and four detection-level species-specific labeling primers were analyzed by colloidal gold-based test strip assay. Moreover, simultaneous detection of fox and pig meat on a double-test line strip was feasible. The CPA strip assay indicated a lower amounts sensitivity of 0.3 ng DNA when one targeted species was tested and a detection limit of 1% when arctic fox meat was detected in the meat mixtures. Using a minimal set of primers, this study provides a promising tool for detecting the species of different types of meat using a constant temperature amplification technology.
Article
Food fraud and species substitution are topical issues that must be addressed for quality and safety purposes and to maintain consumers’ trust. DNA-based methods are considered as the reference approach for species identification, but they are usually limited to a few species only. In this study, the performance of a commercial NGS solution has been evaluated as an untargeted tool to identify meat species. Tested on pure meat samples, all species were correctly identified including several exotic species, and close-related species were correctly differentiated. Species were successfully detected and identified in mixtures down to 1% (w/w). The reliability of the tool was further confirmed on several proficiency test samples, and promising quantification data were obtained. Finally, 45 ground meat samples sourced from local European and Asian markets were analyzed and 18% of them showed cases of adulteration with undeclared meat species.
Article
Authentication of food product ingredients by food control agencies relies on analytical laboratory methods for animal species identification. Such methods must be validated if their results are to be used as acceptable evidence in a court of law. Following the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), we carried out a validation process for the Low Cost and Density (LCD) Array (MEAT 5.0 version) kit for food forensics based on a DNA biochip technology that simultaneously detects 24 animal species. Mixtures of the animal species the kit is designed to detect were prepared at different concentrations and tested on raw/pasteurized, and heated meat and milk matrices. The LCD Array showed high specificity and high sensitivity and it appeared to be robust and repeatable. As such, it provides a useful tool for detecting common adulterants in food items of animal origin.
Article
Full-text available
In recent years a significant increase of food fraud has been observed, ranging from false label claims to the use of additives and fillers to increase profitability. Recently in 2013 horse and pig DNAs were detected in beef products sold from several retailers. Mass spectrometry (MS) has become the workhorse in protein research, and the detection of marker proteins could serve for both animal species and tissue authentication. Meat species authenticity is performed in this paper using a well-defined proteogenomic annotation, carefully chosen surrogate tryptic peptides and analysis using a hybrid quadrupole-Orbitrap MS. Selected mammalian meat samples were homogenised and proteins were extracted and digested with trypsin. The samples were analysed using a high-resolution MS. Chromatography was achieved using a 30-min linear gradient along with a BioBasic C8 100 × 1 mm column at a flow rate of 75 µl min(-1). The MS was operated in full-scan high resolution and accurate mass. MS/MS spectra were collected for selected proteotypic peptides. Muscular proteins were methodically analysed in silico in order to generate tryptic peptide mass lists and theoretical MS/MS spectra. Following a comprehensive bottom-up proteomic analysis, we detected and identified a proteotypic myoglobin tryptic peptide (120-134) for each species with observed m/z below 1.3 ppm compared with theoretical values. Moreover, proteotypic peptides from myosin-1, myosin-2 and β-haemoglobin were also identified. This targeted method allowed comprehensive meat speciation down to 1% (w/w) of undesired product.
Article
Full-text available
• An overview is presented on the global human usage of unconventional animal species (ungulates, rodents, rabbits and hares, kangaroos, reptiles and bats) derived either from wild harvesting or farming • The nutritional value of these species is discussed, focusing on their potential to contribute to food security and to address the protein requirements of a growing population. • The challenges and opportunities arising from the commercial use of these animals are highlighted, as are the problems faced with overexploitation of certain wild species. • Of the species addressed, the rodents appear to present great potential for becoming large commercial commodities for food use.
Article
Full-text available
Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.
Article
Full-text available
Considering that the authentication of food contents is one of the most important issues for the food quality sector, and given the increasing demand for transparency in the meat industry followed the horsemeat scandal in Europe, this study investigates processed-meat products from Italian markets and supermarkets using the mitochondrial cytochrome b gene qualitative PCR identification system in order to verify any species substitution or mislabeling. The results revealed a high substitution rate among the meat products, highlighting a mislabeling rate of 57 %, and consequently, considerable discordance with the indications on the labels, which raises significant food-safety and consumer-protection concerns.
Article
Full-text available
ABSTRACT The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction (PCR) have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.
Article
Full-text available
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 falsi-fication 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 apprehen-sion 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 superi-or 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. Keywords Food forgery . Lipid . Protein-and DNA-based biomarkers . Multiplex PCR Introduction
Article
Full-text available
Inter-species and intraspecific variations in mitochondrial DNA (mtDNA) were observed in a bioinformatics analysis of the mitochondrial genomic sequences of 11 animal species. Some highly conserved regions were identified in the mitochondrial 12S and 16S ribosomal RNA (rRNA) genes of these species. To test whether these sequences are universally conserved, primers were designed to target the conserved regions of these two genes and were used to amplify DNA from 21 animal tissues, including two of unknown origin. By sequencing these PCR amplicons and aligning the sequences to a database of non-redundant nucleotide sequences, it was confirmed that these amplicons aligned specifically to mtDNA sequences from the expected species of origin. This molecular technique, when combined with bioinformatics, provides a reliable method for the taxonomic classification of animal tissues.
Article
Full-text available
Economically motivated adulteration (EMA) of food, also known as food fraud, is the intentional adulteration of food for financial advantage. A common form of EMA, undeclared substitution with alternative ingredients, is usually a health concern because of allergen labeling requirements. As demonstrated by the nearly 300,000 illnesses in China from melamine adulteration of infant formula, EMA also has the potential to result in serious public health consequences. Furthermore, EMA incidents reveal gaps in quality assurance testing methodologies that could be exploited for intentional harm. In contrast to foodborne disease outbreaks, EMA incidents present a particular challenge to the food industry and regulators because they are deliberate acts that are intended to evade detection. Large-scale EMA incidents have been described in the scientific literature, but smaller incidents have been documented only in media sources. We reviewed journal articles and media reports of EMA since 1980. We identified 137 unique incidents in 11 food categories: fish and seafood (24 incidents), dairy products (15), fruit juices (12), oils and fats (12), grain products (11), honey and other natural sweeteners (10), spices and extracts (8), wine and other alcoholic beverages (7), infant formula (5), plant-based proteins (5), and other food products (28). We identified common characteristics among the incidents that may help us better evaluate and reduce the risk of EMA. These characteristics reflect the ways in which existing regulatory systems or testing methodologies were inadequate for detecting EMA and how novel detection methods and other deterrence strategies can be deployed. Prevention and detection of EMA cannot depend on traditional food safety strategies. Comprehensive food protection, as outlined by the Food Safety Modernization Act, will require innovative methods for detecting EMA and for targeting crucial resources toward the riskiest food products.
Article
Full-text available
Background Wild animals’ meat is extensively consumed in South Africa, being obtained either from ranching, farming or hunting. To test the authenticity of the commercial labels of meat products in the local market, we obtained DNA sequence information from 146 samples (14 beef and 132 game labels) for barcoding cytochrome c oxidase subunit I and partial cytochrome b and mitochondrial fragments. The reliability of species assignments were evaluated using BLAST searches in GenBank, maximum likelihood phylogenetic analysis and the character-based method implemented in BLOG. The Kimura-2-parameter intra- and interspecific variation was evaluated for all matched species. Results The combined application of similarity, phylogenetic and character-based methods proved successful in species identification. Game meat samples showed 76.5% substitution, no beef samples were substituted. The substitutions showed a variety of domestic species (cattle, horse, pig, lamb), common game species in the market (kudu, gemsbok, ostrich, impala, springbok), uncommon species in the market (giraffe, waterbuck, bushbuck, duiker, mountain zebra) and extra-continental species (kangaroo). The mountain zebra Equus zebra is an International Union for Conservation of Nature (IUCN) red listed species. We also detected Damaliscus pygargus, which is composed of two subspecies with one listed by IUCN as ‘near threatened’; however, these mitochondrial fragments were insufficient to distinguish between the subspecies. The genetic distance between African ungulate species often overlaps with within-species distance in cases of recent speciation events, and strong phylogeographic structure determines within-species distances that are similar to the commonly accepted distances between species. Conclusions The reliability of commercial labeling of game meat in South Africa is very poor. The extensive substitution of wild game has important implications for conservation and commerce, and for the consumers making decisions on the basis of health, religious beliefs or personal choices. Distance would be a poor indicator for identification of African ungulates species. The efficiency of the character-based method is reliant upon availability of large reference data. The current higher availability of cytochrome b data would make this the marker of choice for African ungulates. The encountered problems of incomplete or erroneous information in databases are discussed.
Article
Full-text available
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 MEATspecies 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. KeywordsDNA biochip–PCR–Meat products–CarnoCheck–LCD Array
Article
Full-text available
We have reviewed the current pig (Sus scrofa) genomic diversity within and between sites and compared them with human and other livestock. The current Porcine 60K single nucleotide polymorphism (SNP) panel has an average SNP distance in a range of 30 - 40 kb. Most of genetic variation was distributed within populations, and only a small proportion of them existed between populations. The average heterozygosity was lower in pig than in human and other livestock. Genetic inbreeding coefficient (F(IS)), population differentiation (F(ST)), and Nei's genetic distance between populations were much larger in pig than in human and other livestock. Higher average genetic distance existed between European and Asian populations than between European or between Asian populations. Asian breeds harboured much larger variability and higher average heterozygosity than European breeds. The samples of wild boar that have been analyzed displayed more extensive genetic variation than domestic breeds. The average linkage disequilibrium (LD) in improved pig breeds extended to 1 - 3 cM, much larger than that in human (~ 30 kb) and cattle (~ 100 kb), but smaller than that in sheep (~ 10 cM). European breeds showed greater LD that decayed more slowly than Asian breeds. We briefly discuss some processes for maintaining genomic diversity in pig, including migration, introgression, selection, and drift. We conclude that, due to the long time of domestication, the pig possesses lower heterozygosity, higher F(IS), and larger LD compared with human and cattle. This implies that a smaller effective population size and less informative markers are needed in pig for genome wide association studies.
Article
Full-text available
A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.
Article
Full-text available
Part of our work aims at studying the modifications that proteins suffer in foods and use them as markers to estimate the origin and history of the product. Proteomics is a powerful approach to do this: comparison of the two-dimensional (2-D) maps of the intact and treated samples would permit to identify marker spots so that in the future it may be possible to estimate the treatment a foodstuff has suffered by examining its 2-D protein map or just the selected markers. This work summarizes some of our previous studies showing the application of proteomics to the (i) identification of species and muscle tissues, (ii) characterization of post-mortem changes in arctic and tropical species, and (iii) study of the effect of some additives during the processing of fish muscle.
Article
The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.
Article
Strong anthropogenic impact has caused 28 of the currently recognized 55 species of deer (Cervidae) to be listed on the IUCN Red List. Particular threats to vulnerable species include habitat deterioration and hybridization with alien, introduced species. The scarcity of many species has severely hampered genetic analyses of their populations, including the detection of loci for cross-species amplification. Because deer antlers are shed and re-grown annually, antlers offer the possibility for non-invasive genetic sampling of large individual numbers, and may provide material for reference genotyping from historical samples stored in zoos, museums and trophy collections of rare and extinct species/populations. In this paper, we report cross-species amplification of 19 nuclear microsatellite loci and the amplification of 16S mtDNA for barcoding from nearly a third of all deer species worldwide based on high quality DNA extracted from antler bone up to 40 years old. Phylogenetic analysis based on mtDNA of seventeen species and five subspecies corroborate previously published phylogenetic data, thus confirming the specific resolution of the DNA extraction methodology.
Article
Due to their high market value, meat products are often targets for species substitution and adulteration. DNA-based methods are recognized as the most appropriate means to detect such fraudulent practices, however, these have not been extensively employed for the authentication of meat products available in South Africa. The aim of this study was to utilize a variety of molecular techniques to evaluate the extent of meat product mislabelling prevailing on the local market. A total of 139 processed meat products (minced meats, burger patties, deli meats, sausages and dried meats) were collected from retail outlets and butcheries in South Africa. The enzyme-linked immunosorbent assay (ELISA) was employed for the detection of undeclared plant proteins (soya and gluten) in the samples. A commercial DNA-based LCD array was used to screen the samples for the presence of 14 animal species, the results of which were confirmed by species-specific polymerase chain reaction (PCR) and in some cases also DNA sequencing. The results revealed that 95 of 139 (68%) samples contained species which were not declared on the product labelling, with the incidence being highest in sausages, burger patties and deli meats. Soya and gluten were identified as undeclared plant proteins in a large number of samples (>28%), while pork (37%) and chicken (23%) were the most commonly detected animal species. Unconventional species such as donkey, goat and water buffalo were also discovered in a number of products. Overall, this study confirmed that the mislabelling of processed meats is commonplace in South Africa and not only violates food labelling regulations, but also poses economic, religious, ethical and health impacts.
Article
Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers’ perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories. Figure A broad multiplex real‐time PCR method for the detection and identification of GMOs
Article
A novel method is presented here as an analytical tool for food control and authentication of dairy products manufactured from the milk of cow, sheep, goat, and buffalo. The method is based on multiplex polymerase chain reaction (PCR) of species-specific mitochondrial DNA (mtDNA) targets followed by fragment size analysis by capillary electrophoresis. The method includes (a) simultaneous detection of four species, (b) internal control for DNA extraction and PCR, (c) mtDNA as a target for PCR, (d) amplicons of <200 bp, and (e) flexibility in the electrophoresis and fragment size detection method. Species identification proved to be straightforward, efficient, sensitive, and robust. The method is sensitive to an at least 1% (v/v) relative proportion of milk in binary mixtures. A survey of commercial products showed that 12.5% failed to conform to the description of the contents, by either the introduction or absence of listed species, thus demonstrating the relevance of this type of testing.
Article
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.
Article
Proper labelling of meat products is important to help fair-trade, and to enable consumers to make informed choices. However, it has been shown that labelling of species, expressed as weight/weight (w/w), on meat product labels was incorrect in more than 20% of cases. Enforcement of labelling regulations requires reliable analytical methods. Analytical methods are often based on protein or DNA measurements, which are not directly comparable to labelled meat expressed as w/w. This review discusses a wide range of analytical methods with focus on their ability to quantify and their limits of detection (LOD). In particular, problems associated with a correlation from quantitative DNA based results to meat content (w/w) are discussed. The hope is to make researchers aware of the problems of expressing DNA results as meat content (w/w) in order to find better alternatives. One alternative is to express DNA results as genome/genome equivalents.
Article
Fatty acid (FA) composition of intramuscular fat (IMF) in M. Longissimus dorsi (LD) was measured in 72 steers from Angus (A), Charolais×Angus (CHA×A) and Holstein Argentine (HA) breeds. The steers were allotted to four dietary treatments of six animals each: T1, steers grazed on pasture; T2, steers supplemented with cracked corn grain (0.7% of live-weight) daily and free access to pasture; T3, steers supplemented with cracked corn grain (1% of live-weight) daily and free access to pasture; and T4, feedlot (concentrate based on corn, alfalfa hay and soybean meal without access to pasture). At slaughter weight, samples of LD at the 11th rib were used for intramuscular lipid analysis. The diet was shown to be more important than breed in determining FA composition. Pasture beef had higher percentages of saturated fatty acids (SFA), n-3 polyunsaturated fatty acids (PUFA) and conjugated linoleic acid (CLA) and lower percentages of IMF, monounsaturated fatty acids (MUFA), n-6 PUFA and n-6/n-3 ratios than feedlot beef. HA beef presented lower percentages of SFA and more MUFA with a higher n-6/n-3 ratio than A and CHA×A. Comparing grass and feedlot beef the amounts of FA in muscle (mg/100g) were, respectively 18:3 n-3 (44 vs. 11mg), CLA (20 vs. 12mg), 20:5 n-3 (20 vs. 11mg), 22:5 n-5 (20 vs. 11mg), 22:6 n-3 (12 vs. 6mg) and n-3 PUFA (84 vs. 32mg). Feedlot beef has more SFA (1372 vs. 1081mg), MUFA (1574 vs. 1078mg), PUFA (350 vs. 227mg) and n-6 PUFA (318 vs.143mg).
Article
A rapid real-time polymerase chain reaction (PCR) technique using SYBR Green detection system, has been developed for the quantification of red deer, fallow deer, and roe deer DNAs in meat mixtures. The method combines the use of cervid-specific primers that amplify a 134, 169, and 120bp of the 12S rRNA gene fragment of red deer, fallow deer and roe deer, respectively, and universal primers that amplify a 140bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The C(t) (threshold cycle) values obtained with the 18S rRNA primers are used to normalize those obtained from each of the cervid-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat treated binary mixtures of red deer, fallow deer or roe deer meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target cervid DNAs in the range 0.1-0.8%, depending on the species and treatment of the meat samples analyzed.
Article
The present work is focused on the use of the relative amounts of carnosine and anserine for detecting the presence of proteins of mammalian origin in feeds for ruminants. The methodology is cheap and simple and may serve for the rapid screening of feeds. Animal protein additions to feeds as low as 0.5% can be easily detected based on the cation exchange HPLC determination of the carnosine and/or anserine dipeptides. Furthermore, the molar ratio of these dipeptides was found to be characteristic of the animal species and could be used to discriminate between mammalian and non-mammalian species eventually added to feeds. Feeds having carnosine/anserine molar ratios higher than 0.3 were strongly suspected of containing banned mammalian proteins. Those suspicious feeds might be later confirmed by more specific techniques. The exact source of feeds containing mixtures of animal proteins from different species could not be identified by this method even though the method was able to detect its mammalian origin.
Article
Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.
Article
The Barcode of Life Data System (bold) is an informatics workbench aiding the acquisition, storage, analysis and publication of DNA barcode records. By assembling molecular, morphological and distributional data, it bridges a traditional bioinformatics chasm. bold is freely available to any researcher with interests in DNA barcoding. By providing specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well positioned to support projects that involve broad research alliances. This paper provides a brief introduction to the key elements of bold, discusses their functional capabilities, and concludes by examining computational resources and future prospects.
Article
The present work investigates the possibility of constructing a multivariate calibration model to predict the composition of ground meat with respect to animal species, based on intensity profiles from isoelectric focusing of water-soluble proteins in mixtures of ground meat. Samples containing various amounts of beef, pork and turkey meat were analyzed by isoelectric focusing in immobilized pH-gradients. The gels were photographed and the images transferred to a digital format. The digital images were processed in order to reduce background and optimize signal strength. Resulting gel profiles were analyzed by multivariate regression allowing the determination of sample composition with prediction errors close to 10%.
Article
In this overview, different meat authenticity issues are presented, as well as a wide variety of methods available for meat authentication. Unlike chromatographic, traditional gel electrophoretic, or immunological methods, which have been routinely used in analytical laboratories, the application of capillary electrophoresis (CE) is relatively new in solving meat authentication issues. Several unique CE applications based on meat protein fingerprinting are discussed for the analysis of meat species in unheated meat products. For protein data interpretation, pattern recognition is used to account for the natural variability present within the same meat species. While gel DNA-based methods are widely used for determining meat species in heat processed products, few DNA-based methods utilizing CE have been reported. Moreover, the methods reported are qualitative or semiquantitative. Thus, the need for quantitative competitive PCR CE methods in the determination of meat species is addressed. For the determination of meat extenders, CE methods were either protein-based or based on specific markers. Polyphenols are used as specific markers for soy detection and hydroxyproline is used as a specific marker for collagen determination. Finally, the potential of electrophoretically mediated miroanalysis (EMMA) for the detection of meat that may have been previously frozen and retailed as "fresh" is highlighted.
Article
The design of real-time polymerase chain reaction (PCR) assays for the detection of meat in processed products has focused on using small amplicons, often to the detriment of specificity. However, the relationship between amplification rates and the amplicon size for processed meat products has yet to be determined. To investigate this relationship, real-time PCR assays were designed to give a series of amplicons of increasing size. These assays were then used to assess amplification rates, in relation to amplicon size, in processed meat matrices. Although the most sensitive assays were those that used the smallest amplicons, amplification was still observed using amplicons of 351 base pairs for highly processed samples. It was found, therefore, that although in general, amplicons should be as small as possible, larger amplicons give efficient amplification and that small amplicons should not be chosen if they compromise assay specificity.