Cloth-based hybridization array system for expanded identification of the animal species origin of derived materials in feeds

A cloth-based hybridization array system (CHAS) previously developed for the detection of animal species for which prohibited materials have been specified (cattle, sheep, goat, elk, and deer) has been expanded to include the detection of animal species for which there are no prohibitions (pig and horse) in Canadian and American animal feeds. Animal species were identified by amplification of mitochondrial DNA sequences by PCR and subsequent hybridization of the amplicons with an array of species-specific oligonucleotide capture probes immobilized on a polyester cloth support, followed by an immunoenzymatic assay of the bound PCR products. The CHAS permitted sensitive and specific detection of meat meals from different animal species blended in a grain-based feed and should provide a useful adjunct to microscopic examination for the identification of prohibited materials in animal feeds.     

Monoclonal antibodies against troponin I for the detection of rendered muscle tissues in animal feedstuffs

Regulatory controls to prevent the spread of BSE have prohibited the use of certain animal proteins in feed in several countries. Accurate analytical methods for detecting prohibited material in feedstuffs are needed to ensure compliance with the new regulations. Six IgG class monoclonal antibodies (MAbs) against troponin I (TnI), a thermostable marker protein, have been developed for the detection and differentiation of rendered muscle tissue in animal feed. MAbs 1F9, 2G3 and 7F7 reacted to TnI of all species, including mammalian, poultry and fish, while MAbs 7A12 and 8A12 recognized only mammalian TnI (porcine, bovine, ovine, equine, and deer). MAb 2A8 was able to differentiate TnI of ruminant origin (bovine, ovine and deer) from other species. Three indirect enzyme-linked immunosorbent assays (ELISAs) employing these MAbs were developed for the determination of animal muscle, mammalian muscle or ruminant muscle in animal feeds.     

Detection and analysis of animal materials in food and feed

Bovine spongiform encephalopathy (BSE) belongs to a group of progressively degenerative neurological diseases known as transmissible spongiform encephalopathies (TSEs) associated with a variant form of Creutzfeldt-Jakob disease in humans. TSEs are fatal diseases caused by prions (proteinaceous infectious particle) and are characterized by an incubation period that may range from several months to several years, depending on the host. Because BSE is spread through animal feed, the main strategy for preventing the establishment and spread of BSE is to prohibit the use of proteins derived from mammalian tissue in feed for ruminant animals. Enforcement of these regulations relies on the ability to identify the presence of prohibited proteins in ruminant feed. The methods to detect bovine products in rendered and cooked materials are based on analyses of DNA, bone, or protein. In this article, we discuss the current methodology as well as other potentially useful methods of analysis of animal material in food. While methods are generally useful, none specifically distinguish between prohibited bovine material and allowable bovine products, such as milk or blood. Furthermore, all these methods are hampered by the fact that the rendering process involves heat treatment that denatures and degrades proteins and DNA. There is a need for improving existing methods and developing new methods to overcome these two limitations.     

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs.Industrial relevanceThe variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.     

Characterization of a polymerase chain reaction-based approach for the simultaneous detection of multiple animal-derived materials in animal feed

In this study, a polymerase chain reaction (PCR) primer set capable of amplifying a mitochondrial DNA segment of multiple species (cattle, sheep, goats, deer, and elk) whose rendered remains are prohibited from being fed to ruminants was characterized. However, the primer set also amplifies DNA derived from the rendered remains of pigs and horses, which are exempt from the feed ban. PCR amplicons derived from pig DNA have a restriction endonuclease site recognized by Hinf1, while the horse DNA-derived amplicon has a unique restriction endonuclease site recognized by HypCH4III. This "universal" PCR primer produced an amplicon with DNA extracted from dairy feed containing either bovine meat and bone meal or pig blood meal. Enzymatic digestion of the PCR amplicons from these feed samples with Hinf1 resulted in cleavage products only from samples containing pig blood meal. However, Hinf1 digestion of these amplicons was not complete. Further analysis of the pig blood meal with primers specific for bovine or porcine DNA demonstrated the presence of both bovine- and porcine-derived DNA. Enzymatic digestion confirmed these findings. Additional testing was conducted with dry dog food samples labeled as containing either lamb, chicken, turkey, or chicken and fish. The universal PCR primer produced an amplicon only for the dog food containing lamb meal. This paper is the first to describe a simplified approach for the detection of the prohibited species of concern in the feed ban.     

Targets and methods for the detection of processed animal proteins in animal feedstuffs

Bovine spongiform encephalopathy (BSE), or ‘mad cow disease’, is one of several transmissable spongiform encephalopathies (TSEs) known to affect certain mammals and is spread through the ingestion of infected animal tissue. It is believed that the inadvertent contamination of meat and bone meal (MBM) with infected animal tissue and the subsequent use of this material as a feed supplement contributed to the spread of the disease in cattle. As a result, the use of processed animal proteins (PAPs) in animal feeds is regulated in many parts of the world. Although feed testing is the only definitive means to certify compliance, regulatory compliance often relies solely on paper certification. Recently, rapid methods have become available that can be used by regulators to determine compliance during routine inspections. We describe a rapid, immunochromatographic strip test that can detect 0.1% MBM in animal feed. The test takes 15 min to perform and large numbers of samples can be screened for PAPs simultaneously.     

Molecular detection of meat animal species targeting MT 12S rRNA gene

The efficacy of PCR-RFLP analysis of mt 12S rRNA gene in identification of animal species from meat samples of known and unknown origin and adulterated meat samples was evaluated. In PCR, all the samples generated an amplicon of 456 bp. Restriction enzyme digestion of the PCR product with AluI, HhaI, BspTI and ApoI revealed characteristic RFLP patterns. Of the samples of unknown origin few were identified as cattle, few as buffalo and some were admixtures of two, suggesting adulteration. The RFLP pattern of one did not match any of species included in the study, which on sequencing was confirmed as camel meat. Application of this technique on adulterated meat samples could detect both animal species in proportion of 50:50 and 75:25 (except in case of goat+cattle). The technique however could not detect any of the two species when proportion of mixture was 90:10 (except in case of cattle+buffalo).     

Differentiation between Aspergillus flavus and Aspergillus parasiticus from pure culture and aflatoxin-contaminated grapes using PCR-RFLP analysis of aflR-aflJ intergenic spacer

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification.     

Development of immunoassay for detection of meat and bone meal in animal feed

An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130 degrees C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90 degrees C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130 degrees C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.     

食品和饲料中动物源性成分检测技术研究进展

近年来,肉、奶等食品中“掺杂使假”的投诉事件不断上升,疯牛病的传播风险也在不断加大,作为保障食品安全、维护缴费者利益的重要技术手段,食品和饲料中动物源性成分的定性或定量检测技术已成为国内外的研究热点,适用于各种样品、基于各种原理的方法和技术被开发出来。本文综述基于蛋白质和DNA特异性建立的动物源性成分检测技术类型和研究现状,并对其发展趋势进行讨论。     

基于PCR技术的肉类成分溯源鉴定方法研究进展

近几年屡屡曝光的食品安全事故引起了社会的广泛关注,食品安全已经成为社会共同关注的问题,肉类掺假造假现象更是层出不穷,其中用低价鸡肉、鸭肉、猪肉等掺入、冒充牛羊肉成为主要的掺假方式。国内外进行肉类掺假鉴定主要以核酸作为靶标,核酸鉴定也是物种鉴别最常用、最核心的方法,以DNA检测为基础建立起来的DNA条形码、多重PCR、荧光定量PCR、荧光探针等技术也得到空前发展和广泛应用。我国针对动物源性成分检测也制定了相关国家标准和行业标准,但大多现行标准中基于DNA检测建立的PCR技术只能检测单一物种,滞后于技术的发展。目前,基于PCR发展起来的衍生技术凭借其高灵敏度、强特异性和高通量等优势在动物源性成分检测工作中显示出巨大潜力,也是肉类成分鉴定未来的重要方向。本文综述了PCR技术在肉类检测中的研究概况和现行标准的技术概况,以期为肉类成分鉴定研究提供信息。     

Development of primers for detection of meat and bone meal in ruminant feed and identification of the animal of origin

The safe use of cattle feed free from meat and bone meal is an important prerequisite to prevent further spread of bovine spongiform encephalopathy. We designed primers to detect very small amounts of meat and bone meal in ruminant feed. Mitochondrial subunit 8 of the ATP synthase gene was used as a target sequence. PCR-based assays revealed amplification of DNA from mammals, ruminants, and individual species using these primers. The method allowed detection of the presence of meat and bone meal in ruminant feed from 0.1 to 0.01%. Sensitivity and effectiveness of the method for detecting prohibited animal proteins in ruminant feed was evaluated.     

Identification of chicken,duck, pigeon and pig meat by species-specific markers of mitochondrial origin

In the present study, PCR based method for meat species identification of chicken, duck, pigeon and pig was achieved by developing species-specific markers. Using mitochondrial sequences species-specific primers were designed and the sizes of them were 256 bp, 292 bp, 401 bp and 835 bp for chicken, duck, pigeon and pig, respectively. The species-specific PCR products were sequenced to confirm the specificity of the product amplified. These markers were subsequently tested for cross amplification by checking them with beef, mutton, chevon, pork, rabbit, chicken, duck, turkey and pigeon meat. DNA markers developed in this study can help identify the species of fresh, cooked and autoclaved meat of chicken, duck and pigeon and fresh and cooked meat of pig. The process of identification is simple, economical and quick as compared to other methods such as RAPD, PCR-RFLP and sequencing method of species identification.     

A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences

Molecular species detection in food has become common in the last 10 years. The methods are sensitive enough to detect small, but relevant, amounts of one species in composed food. We have developed a strategy for detecting different animal species in food by molecular means. This strategy uses a combination of published PCR systems and new developed PCR primer systems for the detection of porcine, bovine, ovine, avian, cervine and equine DNA by PCR followed by restriction analysis (PCR-RFLP). In some cases, analysis is completed by DNA sequencing. The species detection system includes an amplification control and so is in accordance with the relevant food standards.     

Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy^® Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.     

基于PCR多物种鉴定技术及其在肉类 鉴定中的应用

国内外的肉类掺假由来已久,法律上监管部门对此采取了严格监管措施,技术上应用了感官、显微检测和免疫学等相应的检测技术。近几年来,PCR及其衍生技术的快速发展大大推动了肉类鉴定技术的快速发展,尤其在多物种鉴定技术领域建立了如多重PCR、通用单引物多重PCR、通用引物特异性PCR和PCR-RFLP等多项技术。这些技术的发展为肉类及其制品的检测提供了重要手段,代表了检测技术领域发展的方向,但其也各有其优缺点。本文介绍了上述4种多物种鉴定技术及其在肉类鉴定中的应用,并分析了其优缺点,旨在为物种鉴定方法选择及其研究提供参考。     

Polymerase chain reaction detection of bacterial ribosomal genes from fresh and stored animal feeds

Feed quality can be influenced by both the microbial population levels and types of organisms that become associated with the feed matrices during processing and storage. Evaluation of microbial quality of animal feeds is difficult, because feeds generally contain a diverse bacterial population that can fluctuate widely depending on a variety of factors. Microbial diversity may be investigated in animal feed using the polymerase chain reaction (PCR) and 16S rDNA primers. In this study a bacterial isolation step involving centrifugation was combined with several DNA extraction techniques, and PCR amplicons were visualised on electrophoresis gels. Seven different animal feeds and two commonly used feed ingredients, either fresh or stored for approximately 14 months at ?20 °C, were chosen (18 feed sources in total) to represent a variety of different matrices, concentrations of macronutrients such as protein and fat, and particle sizes. DNA extraction involving polyethylene glycol 8000 appeared to be the most reliable protocol for the extraction of community DNA for PCR analysis of feeds. The majority of the feeds (14 out of 18 animal feeds and feed ingredients) examined in this study yielded at least one PCR‐positive replicate (1.1 kb band on gel), whereas no amplified products could be obtained from either of the other two DNA extraction protocols. Although some protocol refinement may be necessary for individual feeds, this approach has the potential to be a reliable method for monitoring microbial diversity changes in feeds and for rapid, simultaneous detection of a wide variety of micro‐organisms in animal feeds during processing and storage. © 2002 Society of Chemical Industry     

Development of a Polymerase Chain Reaction System for the Detection of Dog and Cat Meat in Meat Mixtures and Animal Feed

The identification of species origin of meat represents a considerable problem for food and animal feed analysis. In the present study a PCR‐mediated method for the detection of dog and cat meat was developed. For this the cytochrome b gene sequence of both species was analyzed by restriction fragment length polymorphism (RFLP) analysis. The use of the restriction enzymes Alu I and Hae III yielded specific restriction profiles characteristic for each species. The meat of both species could additionally be differentiated with species‐specific oligonucleotide primers based on specific parts of the cytochrome b gene sequences characteristic for dog and cat. The use of these oligonucleotide primers allowed a direct identification of dog and cat meat in meat mixtures even after heat treatment.     

Development of a real-time PCR protocol for the species origin confirmation of isolated animal particles detected by NIRM

At present, European legislation prohibits totally the use of processed animal proteins in feed for all farmed animals (Commission Regulation (EC) No. 1234/2003–extended feed ban). A softening of the feed ban for non-ruminants would nevertheless be considered if alternative methods could be used to gain more information concerning the species origin of processed animal proteins than that which can be provided by classical optical microscopy. This would allow control provisions such as the ban of feeding animals with proteins from the same species or intra-species recycling (Regulation (EC) No. 1774/2002). Two promising alternative methods, near-infrared microscopy (NIRM) and real-time polymerase chain reaction (PCR), were combined to authenticate, at the species level, the presence of animal particles. The paper describes the improvements of the real-time PCR method made to the DNA extraction protocol, allowing five PCR analyses to be performed with the DNA extracted from a single particle.     

朊蛋白疾病及其肉食品安全的研究进展

英国疯牛病造成了人类疾病传播的潜在危害,牛羊肉等肉食品的安全备受关注。为了避免疯牛病污染的肉品进入食物链,世界多个研究小组对各类肉食品开展了基于疯牛病传播因子安全方面的研究。疯牛病患病牛肉可以直接感染人类并导致发病,阳性病牛应彻底销毁并进行无害化处理,严禁进入食物链。染病的羊肉和鹿肉属于高度危险食品,也应做销毁处理,而猪肉、马肉、兔肉和水牛肉等则相对比较安全。同时,朊蛋白疾病的感染还与人类自身朊蛋白编码基因多态性有关。在严格检疫的同时,培育抗病品种的家畜以及食用不易感的肉类食品成为未来肉食品安全的一种发展趋势。本文总结了疯牛病、痒病等可传播海绵状脑病患病动物的肉用安全性,还分析了疯牛病可能突破种间屏障而造成跨物种传播的可能性,为肉食品安全及检疫提供参考。