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.     

A simple one-step PCR method for the identification between European and American razor clams species

A specific multiplex polymerase chain reaction (PCR) was applied to differentiate samples of razor clams Ensisarcuatus, Ensissiliqua, Ensisdirectus, and Ensismacha. Universal primers were used for the amplification of internal transcribed spacer 1 (ITS-1) in each species. The alignment of the obtained sequences was the basis for the specific design of species-specific reverse primers (ITSArSil-R, ITSDir-R, and ITSMa-R) located in the ITS-1 region. A multiplex PCR using each specific primer together with a common forward primer allowed identification of razor clam species by means of the different sizes of the species-specific amplicons separated in an agarose gel electrophoresis. This work provides a simple, reliable and rapid protocol for the accurate identification of Ensis species. The present methodology can be very useful for traceability of the species and to reinforce labelling regulations.     

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.     

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。      

Polymerase chain reaction assay for identification of chicken in meat and meat products

The aim of this study was to develop polymerase chain reaction (PCR) assay for specific detection of chicken meat using designed primer pair based on mitochondrial D-loop gene for amplification of 442 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The PCR result was further verified by restriction digestion with HaeIII and Sau3AI enzymes for specific cutting site in amplified DNA fragments. The specificity of assay was cross tested with DNA of cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail, where amplification was observed only in chicken without cross reactivity with red meat species. However positive reaction was also observed in quail and turkey. In this study, no adverse effects of cooking and autoclaving were found on amplification of chicken DNA fragments. Thus, the detection limits was found to be less than 1% in admixed meat and meat products. The developed assay was found specific and sensitive for rapid identification of admixed chicken meat and meat products processed under different manufacturing conditions.     

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.     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

An enzyme-linked immunosorbent assay for species identification of raw meat

An enzyme-linked immunoassay has been developed to differentiate between unprocessed beef, sheep, horse, kangaroo, pig and camel meats. Microtitre plates were coated with meat extracts at pH 5.5 and the bound meat proteins reacted with species-specific rabbit antisera. The antisera were prepared by inoculating rabbits with serum from the required species and were purified by affinity chromatography on appropriate immobilised serum columns to remove cross-reacting antibodies. Rabbit anti-bodies bound to the meat proteins were detected using staphylococcal protein A conjugated to horse-radish peroxidase. The assay is able to detect contamination as efficiently as the currently employed Ouchterlony immunodiffusion test. In addition it has the following advantages: (a) testing can be performed in approximately 3 h; (b) less volume of species-specific antisera is required; (c) antisera can be mixed for simple screening procedures; (d) equipment is avilable to semi-automate the assay procedure and the recording and reporting of results; (e) increased sensitivity, in the terms of amount of material required, allows use of simpler sampling techniques.     

Species identification in meat products using real-time PCR.

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

ELISA-test for cooked meat species identification on gelatine and gelatine products

The results of the ELISA-test with a commercial cooked meat species identification kit on gelatine and gelatine containing products can be influenced by different gelatine types and concentrations leading to false positive readings. The test cannot be applied especially to meat products containing relatively high amounts of soluble collagens because their extracts form solid gels in the test wells. In this case the different reagents added will penetrate the gels and react with each other. The kit may not speciate reliablely commercial gelatine in isolation or in products where gelatine is the sole animal protein source.     

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.     

PCR assay for the identification of animal species in cooked sausages

A species-specific PCR assay was developed for the detection of low levels of pork, horse and donkey meat in cooked sausages. Oligonucleotid primers were designed for amplification of species-specific mitochondrial DNA sequences of each species and detected the presence of 0.01 ng of template DNA in water. When applying the assay to DNA extracts from sausages samples that were prepared from binary meat mixtures, it was possible to detect each species when spiked in any other species at the 0.1% level. In conclusion, it can be suggested that this assay can be used to determine mislabelled and/or fraudulent species substitution in comminuted meat products.     

A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods.     

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.     

Improved species identification of raw meat by double sandwich enzyme-linked immunosorbent assay

An improved enzyme-linked immunosorbent assay (ELISA) has been developed to enable differentiation of unprocessed beef, sheep, horse, kangaroo, pig, camel, buffalo and goat meats to less than 1% level of detection. This double sandwich system utilises species-specific capture antibodies raised in sheep or cattle and coated on to microtitre plates. Antibody coated plates are then used to immunoextract soluble protein from prepared meat samples. Bound meat proteins are detected by the addition of species-specific rabbit antisera followed by staphyloccocal Protein A conjugated to horse-radish peroxidase (HRPO), or by antisera directly conjugated to HRPO. Adoption of a capture antibody provides a number of advantages over previously described ELISA systems. Sample preparation is not critical because colour production is approximately constant between sample dilutions of 1000 and 10 g litre^?1. Increased sensitivity and selectivity allows the bulking of samples for screening purposes. In addition, the assay is faster; testing may be carried out in less than 2 h.     

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。     

A novel method for species identification in milk and milk-based products

This study describes a method for species-specific detection of animal DNA from different species (cattle, sheep, goat, water buffalo) in milk and dairy products. A primer set was designed in conserved region on the basis of the alignment of the sequence codifying the genomic kappa-casein gene in order to amplify all four species with a single primer pair. Polymorphisms were detected via minisequencing with extension primers designed in conserved sequences for haplotype determination that allow unambiguous assignment to each species. The method was successfully applied to the detection of raw and pasteurized milk from the four different species considered as well as to cheese products from the retail trade. Estimation of the limit of detection was carried out using a progression of dilutions of genomic DNA as well as DNA isolated from milk of a known number of somatic cells from different species in order to be able to achieve detection rates as low as 0.1% bovine milk mixed with buffalo milk.     

荧光定量PCR方法检测畜肉食品中猪源性成分

目的 建立一种快速、特异、灵敏的猪源性成分检测方法。方法 本研究以猪线粒体12S rRNA基因序列为靶位点设计引物和探针, 进行荧光定量PCR扩增, 建立猪源性成分检测方法; 以常见畜禽肉包括羊肉、牛肉、鸡肉、鹅肉、鸭肉、兔肉、马肉、鹿肉等参考动物物种作特异性检测; 以50 mg/kg羊肉DNA作为稀释液对猪肉DNA进行梯度稀释, 做灵敏度检测。结果 该方法能够有效对猪源性成分进行快速检测, 具有较强的特异性, 灵敏度较高(可达0.1 μg/kg)并且羊肉成分的存在对猪肉灵敏度检测没有影响。结论 该方法特异性强, 灵敏度高, 可以快速、准确检测畜肉食品中含有的猪源性成分。     

肉制品中牛源性成分Taqman荧光定量PCR检测法的建立

本实验根据牛线粒体差异序列设计特异性PCR引物和Taqman探针,并基于16S rDNA内参基因设计通用引物及Taqman探针,绘制并通过标准曲线确定样品中总DNA浓度以及牛源性成分DNA浓度,采用相对定量法测定肉制品中牛源性成分的质量分数。并通过特异性、灵敏度实验,及混合肉样回收率及市售肉制品检测,对本方法进行验证。结果表明,方法特异性强,可对牛肉DNA进行特异性扩增。最低检出限为10 pg/μL,具有较高的灵敏度。并根据回收实验可知,平均回收率为98.62%。可以为肉制品中牛肉含量的测定提供技术参考。