Sandwich ELISA for detection of horse meat in raw meat mixtures using antisera to muscle soluble proteins

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of horse meat (1-50%) in unheated meat mixtures. The assay uses horse-specific antibodies obtained by immunoadsorption of the crude horse antisera onto immobilised sarcoplasmic extracts from chicken, beef and pig to remove cross-reacting antibodies. The purified antibodies bound to a solid support sequester horse muscle soluble proteins from meat mixtures. Further immunorecognition was made with the same antibodies conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of minced beef and pig containing variable amounts of horse meat.     

Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork

A sandwich ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of chicken meat (1-100%) in beef and pork meat mixtures. The assay uses a monoclonal antibody (BC9) specific to a chicken muscle soluble protein to capture this protein from complex meat mixtures. Further immunorecognition of the captured protein was attained with rabbit polyclonal antibodies against chicken muscle proteins (anti-CHSP). A commercial goat anti-rabbit immunoglobulin conjugated to peroxidase was used to detect the anti-CHSP antibodies bound to the chicken protein. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of beef and pork meats containing variable amounts of chicken meat.     

Production of a horse-specific monoclonal antibody and detection of horse meat in raw meat mixtures by an indirect ELISA

A stable hybridoma cell line (DD3) has been produced secreting a monoclonal antibody specific for horse muscle proteins. The DD3 monoclonal antibody (mAb) did not show significant cross-reactivity when tested against beef, chicken, pig and soya proteins or bovine caseins, gelatine and bovine serum albumin. The DD3 mAb was used in an indirect ELISA format for the detection of defined amounts of horse meat (10–500 g kg-¹) in beef meat mixtures. Immunorecognition of monoclonal antibodies adsorbed to horse meat adsorbed onto the ELISA plate was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of the substrate gave clear optical density differences when assaying mixtures of minced beef containing different amounts of horse meat.     

Optimization of emulsion characteristics of beef, chicken and turkey meat mixtures in model system using mixture design

Emulsion pH (pHe), emulsion capacity (EC), emulsion stability (ES), emulsion density (ED) and apparent yield stress of emulsion (raw emulsion, AYSe) and emulsion gel (cooked emulsion, AYSg) of beef, chicken and turkey meats and their mixtures were studied using a model system.

Turkey meat homogenate was found to have higher protein concentration than chicken or beef homogenates. The highest pHe, EC and ES values and the lowest ED and AYSe values were found in chicken meat. However, the highest AYSg value was found in chicken–turkey meat mixture. Generally, the increasing amount of chicken meat in mixtures increased EC and ES, and decreased ED and AYSe values. Also, chicken–turkey meat mixtures had lower ES values than the mixtures containing only chicken or only turkey meat. With beef, the addition of chicken and turkey meats improved emulsion characteristics significantly. Optimum levels of beef, chicken and turkey meats were found to be 0–23%, 9–30% and 53–91% respectively.     

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.     

The detection of chicken meat in meat products by means of the anserine/carnosine ratio

A distinctive difference was found between the ratio of the anserine and carnosine contents (a/c ratio) in beef or pork and of that in chicken/meat. The a/c ratio for beef varies between 0.06–0.2 and for pork between 0.02–0.1 but for chicken meat can reach values as high as 2.2–5.5.The high a/c ratio for chicken meat proved to be sufficient to detect this ingredient at a 5% level in both cooked pork products and 1:1 beef-pork mixtures, this being independent of the heat treatment.     

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.     

A modified indirect ELISA procedure for raw meat speciation using crude anti-species antisera and stabilised immunoreagents

Indirect enzyme-linked immunosorbent assay (ELISA) for species identification of unheated meat materials has been modified to facilitate the use of non-specific (unpurified) anti-species antisera by integral assay inhibition of heterologous cross-reactivity. Effective ELISA treatments of three antisera (identifying beef, horse and pig) and four commercial products (identifying beef, horse, pig and sheep/goat) were determined using chequer-board microtitration assays against pure species antigens. Subsequent tests with aqueous meat extracts gave consistent absorbance differences between the homologous (high colour) and heterologous (low colour) responses and provided reliable, accurate species identification without the extensive prior purification of antisera (i.e. by affinity chromatography) previously required. This modified ELISA enabled efficient use of stabilised anti-species sera discs, prepared initially for application in a simplified agar-gel immunodiffusion (AGID) test; i.e. one disc (20 μl freeze-dried antiserum) for testing two meat samples by the prescribed AGID made sufficient treated solution to test 50 meat samples on microtitre plates. Authentic species meat extracts are also stabilised on discs for convenience (reference responses and routine controls). ELISA in this form is a practical alternative to AGID screening tests or can be used in parallel back-up tests providing sensitive results in less than 3 h on a large or small scale.     

Production and partial characterization of monoclonal antibodies specific to cooked poultry meat

Monoclonal antibodies (MAbs) specific to cooked poultry muscle proteins have been developed for the detection of poultry adulterants in cooked mammalian meat. Saline (0.85% NaCl) extract of heat-treated (100 °C, 15min) chicken muscle proteins was used to immunize mice for MAb development. The specificity of MAbs was tested against chicken antigen and protein extracts from seven other meat species (pork, beef, lamb, deer, horse, turkey and duck) by indirect enzyme-linked immunosorbent immunoassay (ELISA). The immunogenic components in the poultry protein extracts were determined by SDS-PAGE followed by immunoblotting. A total of six hybridoma cell lines that secrete IgG class MAbs have been developed: MAbs 3E12 and 1A5 were able to distinguish between cooked poultry and mammalian meats, MAbs 9C6 and 6F7 reacted strongly with cooked chicken only, and MAbs, 5D2 and 6G8, reacted with both cooked turkey and chicken but not other species. All six MAbs demonstrated a proportional increased ELISA response to respective adulterated poultry samples in pork over a 0-100% range of aduleration.     

The kinetics of cooked meat haemoprotein formation in meat and model systems

The rate of cooked meat haemoprotein formation (measured as the rate of loss of myoglobin solubility) was found, at least initially, to obey first order kinetics in meat, aqueous muscle extracts and mixtures of myoglobin and bovine serum albumin. In meat at 60 °C the rate was dependent on the species, (the pigment was formed significantly faster in lamb m. longissimus dorsi than in beef m. longissimus dorsi) and anatomical location (cooked meat haemoprotein was formed in beef m. 1. dorsi about twice as rapidly as in both beef shin and chuck (shoulder) muscle of similar pH). The rate of formation was similar in aqueous muscle extracts to that found in meat and in these systems increased with decreasing pH. The activation energies for all beef systems studied were similar and typical of those associated with protein denaturation (˜300 KJ mol⁻¹); however, that from lamb appeared to be lower (˜200 KJ mol⁻¹). The problems of using colour as an index of temperature reached, either for microbial safety (E. Coli 0157:H7 destruction) or quality are discussed in the light of these results.     

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.     

2013年苏州地区肉及其制品掺假情况调查

了解苏州地区肉及其制品的掺假情况,通过对肉类种源与标签明示肉源进行比对,鉴别摻假食品,为加强食品标签管理提供依据。方法 运用自建的动物源性食品种源判定Taqman实时荧光PCR检测体系对苏州地区的肉及其制品进行种源判定,与标签明示肉源进行比对,鉴别摻假食品。结果 本次调查共检验涉及32个生产单位的90份样品,总不符合率为25.6%(23/90)。检测的44份牛肉及其制品中有12份与标签不符,8份用猪肉部分替代牛肉,1份以鸭肉部分代替牛肉进行销售;此外有3份不含有牛肉成分,存在猪、鸡、鸭源性肉类之外的肉类成分。共检测羊肉及其制品16份,有2份用鸭肉代替羊肉出售,3份羊肉样品中掺入了部分猪成分,其中1份样品还存在单个样品掺杂两种外源肉类的现象(猪源性和鸭源性)。检测猪肉及其制品19份,其中2份样品含有标签未注明的鸡肉成分。在所检测的11份混合肉类样品中有4份成分与标签不符,主要是以廉价的鸡肉取代/部分取代相对高价的牛肉和猪肉。结论 肉制品掺假情况明显,用猪肉、鸭肉部分代替牛肉和羊肉仍是主要的掺假手段,牛肉掺假样品主要是熟制牛肉制品,而火锅食用羊肉卷样品则是羊肉掺假高危品,开展肉制品摻假检测对规范肉制品市场具有积极意义。此外,3份未知种源成分的牛肉样品提示在现有检测基础上还需扩大检测范围,防患于未然。     

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.     

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.     

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.     

Determination of papain in raw meat by immunoassay

An antibody raised to papain, a meat tenderising proteolytic enzyme obtained from papaya (Carica papaya), has been used in the development of an enzyme-linked immunoabsorbent assay (ELISA) for the determination of papain in raw meat. Quantitative determinations of papain up to 4 mg/kg of raw meat have been obtained using standard extracts prepared by the exogeneous addition of papain to raw beef. A sample of commercially treated 'tenderised beef' was shown to contain papain at the level of 0·40 mg/kg. In collaboration with a Public Analyst, a papain immunoassay kit has been used to assay 50 samples of beef bought from retail outlets, with a view to monitoring the use of this tenderiser by the meat industry.     

Detection of meat species using TaqMan real-time PCR assays

Species-specific real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specific primers. For detection purposes, two TaqMan probes were developed; the first was specific to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%.     

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.