Identification of meat species by TaqMan-based real-time PCR assay

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

The detection of horse and donkey using real-time PCR

We have developed real-time PCR assays specific for horse and donkey, applicable to the detection of low levels of horse or donkey meat in commercial products. Primers, designed to the mitochondrial cytochrome b gene, were 3′ mismatched to closely related and other commercial species. Amplification of non-target species DNA was prevented by truncation of primers at the 5′ position, thereby conferring complete specificity. Both assays were highly sensitive and detected the presence of 1 pg of donkey template DNA or 25 pg of horse template DNA when assessed using dilutions of DNA in water. Model food samples, spiked with horse or donkey muscle and commercial products containing horse, were successfully tested for the presence of horse or donkey, demonstrating the applicability of the assays to food products.     

Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods

The method performance characteristics of commercially available PCR kits for animal species identification were established. Comminuted meat products containing different levels of pork were prepared from authentic beef, chicken, and turkey. These meat products were analysed in the raw state and after cooking for 20 min at 200 °C. For both raw and cooked meats, the PCR kit could correctly identify the animal species and could reliably detect the addition of pork at a level below 0.1%. A survey of 42 Turkish processed meat products such as soudjouk, salami, sausage, meatball, cured spiced beef and doner kebap was conducted. Thirty-six samples were negative for the presence of pork (< 0.1%) and four were found to be correctly labelled as containing pork. However, one sausage sample was labelled as containing 5% beef, but beef DNA was not detected and a meatball sample labelled as 100% beef was found to contain chicken. Another turkey meatball sample was predominantly chicken.     

Optimization of multiplex PCR for the identification of animal species using mitochondrial genes in sausages

Detection of species fraud in meat products is very important in order to protect consumers from undesirable adulteration, as well as for the economic, religious and health aspects. The most important reason for verification of the labeling statements is to detect fraudulent substitution of expensive meat components with other cheaper animals or mislabeling. The aim of this study was to develop a multiplex PCR that could be used in the simultaneous identification of multiple meat species. In this study, ten sausages with a minimum beef content of 55 %, from ten different manufacturing companies, and five samples of cow, chicken, goat, camel and donkey raw meats, for the purpose of positive control, were collected from food markets in Tehran, Iran. Total DNA was extracted from each sausage and the raw meats. Primers were selected in different regions of mitochondrial DNA (12S rRNA, cytochrome b and NADH dehydrogenase subunits 2) for identification of meat species. 12S rRNA and NADH dehydrogenase subunits 2 primers generated specific fragments of 183 and 145 bp length, for chicken and donkey, respectively. Three different specific primers were used for amplification of cytochrome b gene in goat, camel and cattle species and amplified species-specific DNA fragments of 157, 200 and 274 bp, respectively. The results proved that half of the specimens were contaminated with chicken meat, and this was greater than the proportion of beef stated on the label, while the other half only had chicken residuals, and no beef content. No contamination was found with goat, donkey or camel meats. These findings showed that molecular methods, such as multiplex PCR, is a potentially reliable, sensitive and accurate assay for the detection of adulterated meat species in mixed meat products.     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

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。     

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.     

Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Sardinian sausage, a typical Italian dry fermented sausage. Samples were taken at different stages, and 112 strains were isolated. The isolates were characterized using the micromethod proposed by Font de Valdez et al. [Font de Valdez, G., Savoy de Giori, G., Oliver, G., & De Ruiz Holgado, A. P. (1993). Development and optimization of an expensive microsystem for the biochemical characterization of lactobacilli. Microbiologie Aliments Nutrition, 11, 215–219]. Schillinger and Lücke’s [Schillinger, U., & Lücke, F. K. (1987). Identification of lactobacilli from meat and meat products. Food Microbiology. (4), 199–208] scheme and the biochemical patterns given by Bergey’s Manual of Systematic Bacteriology [Bergey’s Manual of Systematic Bacteriology (1986). Baltimore: William and Wilkins] were used for preliminary identification. A PCR-based method was then used to confirm the results. LAB were the dominant flora during ripening. They consisted mainly of homofermentative mesophilic rods. Lactobacillus sakei (43,3%), Lactobacillus plantarum (16,6%) and Lactobacillus curvatus (13,3%) were the main isolates. The results of the biochemical identification methods agreed well with those of PCR-based identification (91% agreement).     

Rapid identification of deer products by multiplex PCR assay

Attempts were made to establish one-step multiplex PCR assay for the identification of the widely used species in deer products (sika deer, wapiti, red deer and reindeer). Primers were designed from tandem repeat region of D-loop and well-conserved region of 16S rDNA after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 307 bp in length for sika deer, 307 and 246 bp for wapiti, 272 bp for Tarim red deer, 230 bp for red deer and 141 bp for reindeer, respectively. The detection limit was 0.05 ng for sika deer and wapiti, 0.1 ng for Tarim red deer, 0.5 ng for red deer and 0.02 ng for reindeer. The results demonstrated that the fraudulent phenomenon is epidemic in the substitution of deer products, in especially antler, penis, foetus and tendon products. Hence, this multiplex PCR provided a useful and sensitive technique to identify the sources of deer products.     

PCR技术在肉类掺假检验中的应用进展

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

PCR assay for the detection of Campylobacter in marinated and non-marinated poultry products

During a period of 9 months, 194 marinated and non-marinated poultry products were collected from retail shops in a defined area in Western Finland and tested for Campylobacter spp. using a conventional enrichment culture and Polymerase Chain Reaction (PCR) method. For marinated poultry products, the study involved modification of a commercial DNA isolation method. Using either a conventional culture or PCR method, a total of 25 (12.9%) of all investigated samples were Campylobacter positive. In marinated poultry products, Campylobacter was detected at a prevalence of 21.1% and 9.5% in turkey and chicken products, respectively. In August, there was a peak with 28.9% positive Campylobacter samples. Campylobacter inoculation tests were carried out to test the detection limit of both methods. The PCR method used is faster than microbiological analyses. However, enrichment of the samples is necessary due to the low occurrence of Campylobacter spp. in retail Finnish poultry products.     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

Fructooligosaccharides as a fat replacer in fermented cooked sausages

Fermented cooked sausages with a 50% reduction in pork back fat and addition of 0%, 3%, 6% or 9% of fructooligosaccharides (FOS) were produced and studied during manufacturing and storage. Their production was monitored by physicochemical (pH, water activity, weight loss, proximate composition, colour and texture profiles) and microbiological analysis (aerobic mesophiles, lactic acid bacteria, and total and faecal coliforms). During storage, it was evaluated the sensory properties, stability to lipid oxidation and microbiological safety. The final fat content of the control was 27.54%. F0, F3, F6 and F9 treatments had final fat contents of 17.63%, 17.55%, 17.91% and 17.59%, respectively, representing an approximately 40% reduction in the fat content. The simple reduction in pork back fat without fat substitute adversely affected the technological and sensory properties of the fermented cooked sausages, but the addition of FOS suppressed the defects caused by the fat reduction. The content of FOS did not changed during storage, indicating that this functional prebiotic compound can be used for developing of reduced fat fermented meat products.     

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。     

Application of species-specific PCR for the identification of dried bonito product (Katsuobushi)

The species-specific PCR (polymerase chain reaction) method was developed to identify the species of dried bonito product (Katsuobushi) produced from Euthynnus pelamis, E. affinis, Auxis rochei, A. thazard, and Sarda orientalis. The 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five related Scombridae species were established, and then five pairs of species-specific primer were designed to amplify short length fragments among bonito species. The developed species-specific PCR method was successfully applied to authenticate species of commercial dried bonito products. Hence, this method really provided a useful and academic technique to identify the sources of bonito product.     

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.     

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.     

多重PCR法用于畜肉源性鉴定的研究

根据Genebank中猪、牛、羊的线粒体细胞色素b（Cyt-b）基因序列,设计了猪、牛、羊的通用上游引物和特异性下游引物,通过调节引物比例、反应体系、反应条件以及模板量等优化实验确立了多重PCR检测方法。研究结果显示,该多重PCR方法能够同时扩增样品中猪、牛、羊成分,对混合样品的检出限可达10%;并将该方法用于成都市猪、牛、羊肉的调查研究,得到了理想的结果。      

Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique

Effects of various cooking methods including boiling, roasting, pressure cooking, and pan frying on species determination of beef by PCR was studied. The meat was cooked by boiling at 97.5°C for 140, 200 or 230min, by roasting at 200°C for 80, 120, or 150min or by autoclaving at 120°C for 30, 60, or 90min. The beef sample was pan fried until the meat was acceptable for sensory attributes (45min, meat temperature 115°C, fat temp 173°C) and further cooked until unacceptable. DNA was extracted from samples taken after cooking and a 271bp fragment of the mitochondrial DNA was amplified by PCR. The results indicated that with the exception of pan frying for 80min, beef was determined in all meat samples including the broth and sauce of the roasted meat.     

Detection of soya in sausages by the analysis of sterols

 A method for the detection of soya protein concentrates in sausages, using sitosterol as a marker, was elaborated. The method comprises the rapid extraction of non-saponifiable lipids and their analysis by gas chromatography/mass spectrometry. Levels of 2.5% and 5% soya protein concentrates could be clearly detected by the method. Received: 5 November 1997 / Revised version: 12 January 1998