IDENTIFICATION OF GOAT MEAT USING HIGHLY SPECIES‐SPECIFIC POLYMERASE CHAIN REACTION

ABSTRACT

A highly species‐specific polymerase chain reaction (PCR) assay was developed for the authentic identification of goat. A product of 436 bp was amplified using newly designed primers against mitochondrial D‐loop region. The possibility of cross‐amplification was ruled out by considering as many as 25 other animal species. Suitability of the developed goat species‐specific PCR assay was confirmed for in raw, cooked (60, 80 and 100C for 30 min) and micro‐oven‐processed meat samples (n = 20 each). A sensitivity of 0.1% was established for detection of adulteration and limit of detection of goat DNA was 0.1 pg. This investigation presents a novel PCR assay with its newly designed primers that could be used for the authentic identification of goat species.

PRACTICAL APPLICATIONS

This work details about a novel diagnostic polymerase chain reaction, which could be used for authentic identification of goat species. This approach could be used for the confirmation of goat tissues in raw, cooked, as well as adulterated samples. The developed technique has also applications in the forensic analysis of wild animal‐related disputes, where this work could solve the problem of goat‐related issues.     

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.     

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.     

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.     

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.     

基于COI序列的DNA微条形码技术鉴别熟肉制品中11种肉掺假的研究

建立并优化了基于COI序列的DNA微条形码技术(mini-barcoding)检测熟肉制品中11种常见肉类掺假的方法.样品经超声与真空冷冻干燥处理,提取DNA模板和PCR扩增后,目标扩增物经切胶纯化后进行克隆测序,并将测序结果提交GenBank数据库Blast比对.筛选出适合猪、牛、羊、鸡、鸭、鸽子、马、驴、鹅、兔、鼠...     

Authentication of carabeef (water buffalo, <Emphasis Type="Italic">Bubalus bubalis</Emphasis>) using highly specific polymerase chain reaction

In order to ensure consumer satisfaction and fraud detection, correct identification of meat animal species becomes significant. Buffalo being one of the major meat animal species in Asia, a species-specific polymerase chain reaction (PCR) was developed for the accurate identification of carabeef (water buffalo, Bubalus bubalis) targeting mitochondrial D-loop region. Unique diagnostic PCR developed in this study employed novel primers to yield a 534-bp buffalo-specific PCR product, and chances of cross-amplification were excluded by including as many as 25 animal species. Applicability of PCR was established in raw, cooked (60, 80 and 100 °C for 30 min), autoclaved (121 °C for 30 min) and microoven-processed meats with a sensitivity of detection of 0.1% adulteration (10 pg bubaline DNA). Keeping in view adulteration, socio-economic, religious, quality assurance, forensic and legal issues, the novel buffalo-specific PCR developed in this study was found highly promising in authenticating buffalo meat, ensuring consumer satisfaction and labeling process.     

Detection of roe deer,red deer,and hare meat in raw materials and processed products available in Poland

To allow detection of meat from the most popular game species in Poland, we developed a PCR-based method for identification of roe deer (Capreolus capreolus), red deer (Cervus elaphus), and hare (Lepus europaeus). The designed primers were based on the noncoding, mitochondrial D-loop region. Amplicon sizes ranged from 116 to 255 bp. The primers exhibited no cross-reactivity with the DNA from common slaughter and other game species. The detection limit of the assay was established to be below 0.001 % in raw red deer (C. elaphus) and hare (L. europaeus) meat, and below 0.01 % in raw roe deer (C. capreolus) meat, whereas <0.5 % of hare and red deer meat in processed samples could be detected. The PCR-based assay was used for authentication of 17 samples of raw game meat and 32 samples of game meat-containing products available in Polish markets. Analysis of all tested raw meat and processed products revealed the presence of DNA of investigated species in concordance with producers’ declarations.     

TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget

This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R² = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.     

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.     

IDENTIFICATION OF POULTRY MEAT FROM PORK AND BEEF ON THE BASIS OF THE TITIN PEVK REGION USING PCR

The possibility of distinguishing three kinds of meat species (chicken, porcine and bovine) alone or in two-component meat mixtures was investigated using the polymerase chain reaction. The contribution of each component in the meat mixtures ranged from 1 to 100%. The identification of meat was performed on the basis of the sequence coding the PEVK region of titin and by employing polymerase chain reaction. DNA was isolated from raw, fresh and chilled meat. The data presented in this study suggest that it is possible to detect chicken meat, pork and beef in meat mixtures on the basis of PEVK region by using PCR.

PRACTICAL APPLICATIONS

The polymerase chain reaction (PCR) is a precise and quick technique that has many practical applications. Using PCR with primer sets designed on the basis of the PEVK region present in titin can be a convenient tool for species identification, especially for chicken meat mixed with pork and beef meat in two-component meat mixtures. Reliable and sensitive methods for species differentiation can give consumers confidence about authentic meat product composition.     

Effect of Amplicon Length in Propidium Monoazide Quantitative PCR for the Enumeration of Viable Cells of Salmonella in Cooked Ham

Real-time PCR-based methods have been frequently used to detect and enumerate foodborne pathogens. However, these techniques have a major drawback since they cannot differentiate between DNA from live and dead cells. In this study, we developed a propidium monoazide (PMA)-based PCR method to detect and enumerate viable Salmonella cells in the presence of high number of dead cells (up to 10⁸ CFU/g) in cooked ham. Three different specific PCR targets differing in length (95, 285, and 417 bp) were tested. We found that the inhibition effect was dependent on the PCR amplification product length, and only the longer product achieved suppression of 10⁸ CFU/g of heat-killed cells. SYBR^® Green and TaqMan^® chemistries were compared to develop a highly efficient PMA-quantitative PCR system targeting the 417-bp fragment. Both chemistries showed similar detection (10³ CFU/g) and quantification limits (10⁴ CFU/g), but TaqMan^® assay showed higher efficiency (98.6 %) than SYBR^® Green assay (92.8 %). PMA-quantitative PCR assay developed is a rapid method for the selective detection and enumeration of viable Salmonella cells with further application in postprocessed meat products and safe shelf-life studies.     

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.     

Canine-Specific PCR Assay Targeting Cytochrome b Gene for the Detection of Dog Meat Adulteration in Commercial Frankfurters

This report described a cytochrome b (cytb)-based polymerase chain reaction (PCR) assay for the detection of canine tissues in commercial frankfurters. Discriminating detection of canine derivatives in processed food products has important application in halal authentication as well as in health, religions, and fare trades. The assay based on a pair of canine-specific primers that targeted a 100 bp region of canine mithochondrial-cytb gene which is present in multiple copies and highly conserved within the same species. The specificity of the assay was tested against dog and eight most common animal meat species as well as five plant species commonly found in frankfurter formulation. The stability and specificity of the assay were verified under different thermal processing conditions under pure and complex matrices. Three commercial brands of chicken and beef frankfurters were tested in triplicate, and specific PCR products were obtained only from deliberately contaminated formulations. The detection limit of the assay was 0.1 % (0.02 ng DNA) of canine meat spiked with other meats in a typical frankfurter formulation. Shorter amplicon length, superior stability, and higher sensitivity of the assay suggested its potential application in the screening of canine-origin biomaterials in processed food products.     

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.     

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.     

Quantitative Detection of Poultry in Cooked Meat Products

ABSTRACT: There is a growing awareness of perceived harm from meat species adulteration, both intentional and accidental. The present study developed a monoclonal antibody (Mab)-based enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chicken and turkey meat adulterated in cooked (100 °C, 15 min) mammalian meat. The specificity of Mab 5D2 to different species (pork, beef, lamb, deer, horse, duck, chicken, and turkey) and tissues (serum, gizzard, heart, and liver) was studied by noncompetitive ELISA. The detection of cooked chicken in beef, and turkey in pork was accomplished by competitive and noncompetitive ELISAs. Both ELISAs were optimized to quantify cooked poultry in red meats. The new Mab-based ELISAs enabled the detection of cooked poultry in red meats at levels as low as 1% (v/v) or better. The correlation ( r > 0.994) between chicken or turkey concentrations and ELISA signals permitted the quantification of poultry adulterants in cooked non-poultry meats.     

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.