Detection of Adulteration of Meat and Meat Products with Buffalo Meat Employing Polymerase Chain Reaction Assay

The primer pair was designed based on mitochondrial d-loop gene for detection of adulteration of buffalo meat in admixed meat and meat products by polymerase chain reaction (PCR) assay. Amplification of 537-bp DNA fragments was observed from buffalo, without any cross-reaction with cattle, sheep, goat, pig, and chicken. The amplification was further confirmed by BamHI restriction enzymes. No adverse effect of processing was found on PCR amplification of buffalo meat DNA extracted from processed meat and meat products, even from meat emulsion autoclaved at 121 °C, 20 psi for 15–20 min. The detection limit for buffalo meat was found to be 1% in the admixed meat and meat products; however, very faint and inconsistent results were obtained in autoclaved meat emulsion at 1% level. The developed PCR assay was found to be specific for buffalo and could be a useful tool for detection of meat adulteration.     

Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisation with vistra green

A rapid and highly specific assay suitable for the routine detection of turkey and chicken in processed meat products has been developed. Based on PCR amplification of species-specific amplicons with rapid visualisation using vistra green, the assay may be completed within 5 h of receipt of sample. DNA was isolated from meat samples by the use of Wizard DNA isolation technology and followed by DNA amplification in the polymerase chain reaction using species specific primers, chicken forward (CF), chicken reverse (CR), turkey forward (TF) and turkey reverse (TR): the production of an amplicon was detected after the end of the PCR in less than 5 min using vistra green and a fluorescence plate reader. The presence of fluorescence denoted the presence of the target species in the sample.     

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.     

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.     

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.     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

An actin gene-related polymerase chain reaction (PCR) test for identification of chicken in meat mixtures

Using the polymerase chain reaction (PCR) and DNA extracted from muscle, a single pair of oligonucleotide primers can yield amplification products from several members of the actin multigene family simultaneously. These multiple PCR products form species-specific “fingerprints” on gel electrophoresis which may be useful for meat authentication. However, for analysis of meat mixtures, the presence of a single band unique to a species would have many advantages over a multi-component fingerprint. A procedure is described in which primers amplify at a single actin gene locus, giving a positive band with DNA extracted from chicken and turkey, but no reaction with duck, pheasant, porcine, bovine, ovine or equine DNA. The chicken signal was clearly detectable with DNA from meat admixtures containing 1% chicken/99% lamb and from meat heat-treated at 120°C. For further discrimination, the chicken PCR product could be differentiated from turkey by restriction enzyme digestion.     

Determination of pig sex in meat and meat products using multiplex real time-PCR

For specific production lines, European retail companies demand exclusively female pork meat. To control the quality of their suppliers the identification and a quantitative detection of the animal sex origin of the meat is therefore of importance for meat processors. To enable a fast and reliable detection of male pig meat, a real time-PCR-system was designed in the present study. This was based on the genes AMEL-X and AMEL-Y. The real time-PCR assay allowed the detection of male pig meat at a concentration of 1% yielding a detection probability of 100% while the detection probability investigating meat samples containing 0.1% male pig meat was 44.4%. The analytic sensitivity of this system was assessed to be <5 pg DNA per PCR reaction. The assessment of the accuracy of the real time-PCR assay to correctly identify sex individuals was investigated with 62 pigs including males (n=29) and females (n=33) belonging to different breeds/lines. With the newly designed test all analysed animals were correctly sexed. No amplification was obtained with cow, goat, sheep, turkey and chicken genomic DNA. The presented assay can be used for sex diagnosis, for the detection of male pig meat and for meat quality control.     

Ready-to-use single tube quadruplex PCR for differential identification of mutton,chicken, pork and beef in processed meat samples

ABSTRACT

A reliable and sensitive identification method is required to tackle food adulteration mainly in meat production. We developed a dry reagent based ready-to-use single tube quadruplex PCR assay for accurate identification of chicken, mutton, beef and pork. The assay was found to be specific and reproducible. Thermo-stability studies of lyophilized PCR master mix were conducted at different temperature and time intervals, which revealed significant stability for 75 days at 4°C and for 60 days at 25°C. The developed assay was shown to be sensitive down to 16 pg DNA per reaction and the detection limit was found to be 0.01% (w/w) of each species. Furthermore, this method has been applied to the analysis of 68 commercial meat products and the results indicated that nine samples contained non-declared meat components. This dry reagent-based quadruplex PCR assay can be utilized to monitor various processed food products and also to maintain quality control in food industries mainly in the resource-limited settings.     

A novel common single primer multiplex polymerase chain reaction (CSP‐M‐PCR) method for the identification of animal species in minced meat

BACKGROUND: Minced meat species adulteration is a severe problem. In this study a common single primer multiplex polymerase chain reaction (CSP‐M‐PCR) method was applied to the identification of five species (chicken, cattle, sheep, pig and horse) in minced meat products. CSP was designed as a common adapter at the 5′‐end of species‐specific reverse primers and employed in giving different length fragments from the respective meat species. RESULTS: Species‐specific DNA fragments could be identified in a single PCR reaction by mixing CSP, Chicken‐R, Cattle‐R, Sheep‐R, Pig‐R and Horse‐R primers in the ratio 1:0.01:0.01:0.01:0.01:0.10 (where ‘1’ represents 0.5 mmol L^?1). The specific DNA fragments could be efficiently amplified by CSP‐M‐PCR with > 5 µmol L^?1 species‐specific primers, except for 50 µmol L^?1 Horse‐R. A detection limit of 0.5 g kg^?1 was determined for both single species of minced meat and all species of five kinds of minced meat. CONCLUSION: The CSP‐M‐PCR method simplified the PCR reaction system and removed the inconsistent amplification efficiency from different primers. This highly sensitive, reproducible and rapid method could potentially be used as a screening or identifying assay to test for the presence of species or ingredients in minced meat and other meat products. Copyright © 2008 Society of Chemical Industry     

基于TaqMan实时荧光PCR检测鲜肉及加工肉制品中的驴源性成分

根据出入境检验检疫行业标准SN/T 3730.4—2013《食品及饲料中常见畜类品种的鉴定方法 第4部分：驴 成分检测 实时荧光PCR法》合成引物和探针,利用TaqMan实时荧光聚合酶链式反应（polymerase chain reaction, PCR）技术检测鲜肉及加工肉制品中的驴源性成分。首先对13 种不同动物鲜肉组织的DNA进行驴源性成分特异 性检测,然后对驴源性DNA模板原液进行梯度稀释,检测方法灵敏度,最后在加工肉制品中检测方法的适用性。 结果表明：本研究建立的方法特异性强,除驴肉外,牛、羊、猪、马、骆驼、鹿、狗、兔、鸡、鸭、鸽子、鹌鹑 12 种动物鲜肉组织均无特异性扩增;方法的灵敏度较高,驴组分DNA的检出限可达100 fg/μL,灵敏度可达0.01%; 方法的适用性较广,可以用于加工肉制品中驴源性成分的检测。     

Investigation of methods to detect mechanically recovered meat in meat products - IV: Immunology

The possibility of using immunological techniques as a method for the detection of mechanically recovered chicken meat in meat products has been investigated in this preliminary study. Antibodies were raised against a low molecular weight fraction (≤ 30 kDa) of chicken bone marrow proteins and an enzyme-linked immunosorbent assay (ELISA) developed. The system was used to test for the presence of mechanically recovered meat (MRM) in a range of product types, from raw chicken meat through to heat processed samples. The results show that it is possible to raise antibodies to chicken bone marrow proteins which show a strong reactivity with chicken and turkey MRM but show little reaction with extracts of MRM and hand deboned meat of other common meat species. However, blood, skin and soya all affected the accuracy of the ELISA.

This study has demonstrated the potential for the use of an immunological procedure as a rapid test for MRM. The selectivity of the antiserum would, however, have to be increased before this procedure could be considered as a suitable technique for the detection of MRM in meat products.     

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.     

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.     

食品中鸡源性成分实时荧光PCR检测方法的建立

目的：建立基于实时荧光聚合酶链式反应技术的食品中鸡源性成分快速检测方法。方法：以鸡线粒体细胞 色素b为目的基因,设计特异性引物和探针,通过特异性、灵敏性实验及模拟混合肉样和市售肉制品检测,对该体 系进行验证。结果：该鸡源荧光聚合酶链式反应检测体系具有很好的特异性及灵敏性,可检测3.5 pg/μL鸡源DNA 的存在;经含鸡源成分的模拟混合肉样检测,证实体系抗干扰能力强;并且通过市售食品检测表明体系可用于定性 加工食品中的鸡源成分。结论：所建立的鸡源引物探针体系具有特异性好、灵敏度高、快速高效等优点,可用于对 食品中鸡源性成分的掺假鉴别。     

Mitochondrial markers for the detection of four duck species and the specific identification of Muscovy duck in meat mixtures using the polymerase chain reaction

A polymerase chain reaction (PCR) assay for the qualitative detection of four duck species in meat mixtures, and a second PCR assay for the specific identification of Muscovy duck, have been developed based on oligonucleotide primers targeting the 12S rRNA mitochondrial gene. The specificity of both assays was tested against a wide range of animal species. The technique was applied to raw and sterilized muscular binary mixtures, with a detection limit that ranged from 0.1% to 1.0% (w/w). The short length (less than 100 bp) of the DNA fragments amplified with these primer pairs was found to be essential for the successful amplification in samples with highly degraded DNA, and consequently, it could be very useful in inspection programmes to enforce labelling regulation of heat and pressure-processed products, for which other methods cannot be applied.     

Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction

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.     

Development and application of highly specific PCR for detection of chicken (Gallus gallus) meat adulteration

In order to prevent fraud in the sale and strengthen quality assurance, authentic identification of chicken meat is essential. In the present investigation, a chicken (Gallus gallus)-specific polymerase chain reaction (PCR) was developed for the unambiguous identification of chicken meat. The PCR assay employs pair of primers designed against chicken nuclear 5-aminolevulinate (ALA) synthase gene. Highly chicken-specific diagnostic amplicon of 288 bp was established upon PCR and was evident in all the nine breeds/strains of chicken species. Sensitivity of PCR in detecting chicken meat adulteration was established to be at 0.1 % in the foreign meat matrix, while limit of detection (LOD) of chicken DNA was 10 pg. Suitability of the developed chicken-specific PCR was validated and confirmed in raw, cooked/heat treated (60, 80, 100, and 121 °C), and micro-oven cooked meat samples. Possibility of cross-amplification of adulterating DNA was excluded by cross-checking the developed PCR assay with several animal and avian species. The PCR assay developed in this study is highly promising for applications involving circumstances that require authentic identification of chicken meat.     

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.