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.     

Monoclonal Antibodies to Porcine Thermal-Stable Muscle Protein for Detection of Pork in Raw and Cooked Meats

Four hybridoma cell lines secreting monoclonal antibodies (MAbs) specific to porcine thermal-stable muscle proteins (TSMPs) were developed. The MAbs reacted with three protein bands (20.5, 22 and 24 kD) from raw pork extract; the protein band (24 kD) which was also present in cooked pork was identified as porcine-specific TSMP. Epitope analysis indicated four MAbs recognized same or closely located antigenic sites on the pork TSMP. The developed MAb-based indirect enzyme-linked immunosorbent assay (ELISA) enabled the detection of pork in raw and cooked heterogeneous meat mixtures as low as 10 g/kg. The curvilinear relations of second-degree polynomial (r² > 0.995) between pork concentrations and ELISA responses enabled quantifying degree of adulteration of pork in meat products.     

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%.     

Selective Inhibition of Lectin Induced Rabbit Erythrocyte Agglutination by Hydrolysates of Skeletal Beef Protein

Soybean lectin (SBL) induced agglutination of rabbit red blood cells (RRBC) was selectively inhibited by partial tryptic hydrolysates of beef protein isolate and all-beef frankfurter. These interactions have been utilized to design a noninstrumental agglutination method to detect nonbeef muscle protein in meat products. The presence of non-beef muscle proteins in cooked sausages is reflected by a reduced level of inhibition established for the all-beef skeletal muscle hydrolysate. The method effectively detected the presence of 10-50% non-beef proteins (soya, pork, pork rind, chicken, turkey and surimi) substituted in all-beef frankfurter.     

The Use of a Poultry-Specific Murine Monoclonal Antibody Directed to the Insoluble Muscle Protein Desmin in Meat Speciation

Monoclonal antibodies were produced using chicken smooth muscle desmin as the immunogen and one antibody, 4B4/B2, was found to be reactive with desmin from chicken, turkey, pheasant and duck but not from lamb, beef and pork. This antibody reacts with poultry desmin from both smooth muscle and skeletal muscle, allowing its use for the detection of poultry meat in other meats. In addition to reacting with intact forms of this cytoskeletal protein (apparent subunit molecular size 54 kDa), 4B4/B2 also reacts with degradation products of desmin (30–40 kDa), thus enhancing its use for speciation purposes. When the antibody is used in an indirect ELISA involving antigen-coated plates, it can detect <0·04 μg ml⁻¹ poultry desmin. As desmin is one of the most insoluble proteins in cells, it is an ideal marker for speciation. Desmin-enriched samples can be produced from chicken meat and chicken/red meat mixtures by differential extraction, culminating with the solubilisation of desmin in 8M urea. These extracts can be analysed using the ELISA system, routinely detecting chicken at the 100 g kg⁻¹ mixed meats level. Alternatively, Western blots of the samples, resolved by 100 g kg⁻¹ SDS PAGE, can be probed with 4B4/B2 allow-ing detection at the 20 g kg⁻¹ mixed meats level. This monoclonal antibody rep-resents a standard reagent of unlimited supply which can, for the first time, detect a defined insoluble protein in poultry speciation assays. Preliminary applications indicate that it is superior to other reagents and demonstrate that desmin has the potential to be a valuable marker for speciation.     

Determination of soybean proteins in commercial heat-processed meat products prepared with chicken,beef or complex mixtures of meats from different species

The addition of foreign proteins (mainly soybean proteins and milk proteins) to heat-processed meat products is a common practice. This work approaches the determination of additions of soybean proteins in heat-processed meat products prepared with chicken meat, beef meat, and complex mixtures of meats from different species (chicken, pork, beef, and turkey) by perfusion reversed-phase high-performance liquid chromatography. The applied method was previously developed for the determination of soybean proteins in pork and turkey meat products but it has never been tested for the determination of soybean proteins in other heat-processed meat products containing other kinds of meats. This paper demonstrates the validity of this method for the detection of soybean proteins in heat-processed meat products containing different varieties of meats and even in the presence of other foreign proteins such as milk proteins. The specificity and existence of matrix interferences have been checked for these samples and accuracy has been evaluated by the comparison of the soybean protein contents determined by the proposed method and the official ELISA method.     

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 and Characterization of Monoclonal Antibodies Specific to Pangasius Catfish, Basa, and Tra

ABSTRACT:  Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra ( Pangasius hypophthalmus ) and basa ( Pangasius bocourti ), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.     

Monoclonal Antibody-based ELISA for Assessment of Endpoint Heating Temperature of Ground Pork and Beef

ABSTRACT: :
An indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb) 2F8 specific to a mammalian muscle protein was developed to evaluate the endpoint heating temperature (EPT) in ground pork and beef. The ELISA response of MAb 2F8 with meat extracts increased rapidly as the EPT increased from 60 to 80 °C and leveled off above 80 °C. Results of immunoblot revealed that the 12 kD antigenic thermo-stable muscle protein, which is common in both pork and beef, can be used as an EPT indicator for heat-processed pork and beef based on changes in the ratio of this protein to total soluble proteins in the meat extract which occurs at the EPT.     

Production and characterization of monoclonal antibodies specific to chicken muscle soluble proteins

Three stable hybridoma cell lines (AH4, BC9 and CF2) have been produced which secrete monoclonal antibodies specific for chicken and turkey muscle proteins. Partial characterization by ELISA and SDS-PAGE immunoblotting indicated that the antibodies failed to cross-react with similar extracts of pork, beef, lamb, horse or rabbit. One of the cell lines (AH4) secreted a monoclonal antibody that was also capable of distinguishing between chicken and turkey by indirect ELISA.     

Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed Products

A monoclonal antibody‐based sandwich enzyme‐linked immunosorbent assay (ELISA) was developed for the sensitive detection of porcine skeletal muscle in raw and heat‐processed meat and feed products. Heat treatment of meat samples up to 132 °C for 2 h did not affect the assay performance. The assay uses a pair of monoclonal antibodies (MAbs 8F10 and 5H9) specific to skeletal muscle troponin I (TnI). MAb 8F10, reacting to mammalian TnI, is the capture antibody and the biotin‐conjugated MAb 5H9, specific to porcine TnI, the detection antibody. The sandwich ELISA is able to detect 0.05% (w/w) of laboratory‐adulterated pork in chicken, 0.1% (w/w) pork in beef mixtures, 0.05% (w/w) pork meal in soy‐based feed, and 1% commercial meat and bone meal (MBM), containing an unknown amount of pork, in soy‐based feed. This new assay provides a rapid and reliable means to detect the contamination of meat and feed products with trace amounts of porcine muscle tissue to ensure product quality and safety.     

Monoclonal antibodies against troponin I for the detection of rendered muscle tissues in animal feedstuffs

Regulatory controls to prevent the spread of BSE have prohibited the use of certain animal proteins in feed in several countries. Accurate analytical methods for detecting prohibited material in feedstuffs are needed to ensure compliance with the new regulations. Six IgG class monoclonal antibodies (MAbs) against troponin I (TnI), a thermostable marker protein, have been developed for the detection and differentiation of rendered muscle tissue in animal feed. MAbs 1F9, 2G3 and 7F7 reacted to TnI of all species, including mammalian, poultry and fish, while MAbs 7A12 and 8A12 recognized only mammalian TnI (porcine, bovine, ovine, equine, and deer). MAb 2A8 was able to differentiate TnI of ruminant origin (bovine, ovine and deer) from other species. Three indirect enzyme-linked immunosorbent assays (ELISAs) employing these MAbs were developed for the determination of animal muscle, mammalian muscle or ruminant muscle in animal feeds.     

Lactate Dehydrogenase Monoclonal Antibody Immunoassay for Detection of Turkey Meat in Beef and Pork

Relative epitope binding positions on turkey muscle LDH of four monoclonal antibodies (MAbs) was determined by comparing the absorbance by sandwich ELISA from different combinations of capture and biotinlabeled detector MAbs. Epitopes for MAbs E6B and B3C were close to each other. Binding of MAb B3C and G4D did not inhibit binding of D5E to LDH, thus a sandwich ELISA using D5E as capture MAb, and biotin-labeled B3C as detector MAb was developed. The LDH MAb sandwich ELISA detected 1% adulteration of turkey breast or thigh muscle in raw beef and pork.     

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.     

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.     

An Enzyme Immunoassay Technique for Detection of Salmonellae in Meat and Poultry Products

A commercially available enzyme immunoassay (ELISA) in which a myeloma protein (MOPC 467) is used for detection of salmonellae was compared with two conventional cultural methods for detection of salmonellae in naturally contaminated meat and poultry products. Products tested included mechanically deboned poultry, chitterlings, poultry carcass rinsings, chicken necks, luncheon loaf emulsion, pork sausage and basturma. There was 100% agreement between ELISA and cultural methods. The ELISA technique is specific and rapid. Identification of Salmonella-contaminated meat and poultry products was accomplished in 2-3 days compared to the 4-6 days required by conventional cultural methods.     

非标记液质联用法测定市售鸡肉和猪肉的蛋白组

采用非标记液质联用法对市售鸡肉和猪肉样品进行蛋白组测定,比较两种肉在新鲜和熬煮后的小肽种类、量的相对变化。结果显示新鲜鸡肉的蛋白主要是大于100kD的蛋白,pI值范围为6～11;而鸡肉汤中蛋白主要是10～150kD,pI值范围为4～9;新鲜猪肉的蛋白主要是大于100kD的蛋白,pI值范围为6～11,与鸡肉相似;而猪肉汤中蛋白主要是10～100kD,pI值范围为3～9。显示了不同肉材质在相似加工中,其蛋白组的变化会有所不同,根据这些不同,提示由蛋白组测试提取出的指标可为肉质量的评价提供新的评价方式。     

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...     

Characterization of new hybridoma clones producing monoclonal antibodies reactive against both live and heat-killed Listeria monocytogenes

ABSTRACT:  The objective of this study is to develop high affinity monoclonal antibody (MAb) probes recognizing all major serotypes of Listeria monocytogenes cells. From 500 candidate hybridoma clones, 2 new monoclonal antibody-producing hybridomas were selected and evaluated. MAbs 22D10 and 24F6 reacted strongly with live cells of most serotypes of L. monocytogenes except 4c and 4e and with some L. innocua strains; MAb 22D10 reacted strongly with both live and heat-killed cells (100 °C for 20 min) of Listeria . Both MAbs 22D10 and 24F6 did not show any cross-reactions with the other non- Listeria G(+) bacteria tested in ELISA. The mixture of EM-7G1 and 22D10 or 24F6 reacted with all 13 major serotypes of live L. monocytogenes except serotype 4c, while none of these 3 MAbs when tested alone did so. MAb 22D10 mixed with 7G1 reacted with all heat-killed L. monocytogenes serotypes except 4c and 4e. In Western blots, MAbs 22D10 and 24F6 reacted with 1 major protein band of 66 kDa in extracts from L. monocytogenes , but with 2 major protein bands of 66 kDa and 76 kDa in extracts from L. innocua . These results suggest that MAbs 22D10 and 24F6 have high affinity for 11 of 13 serotypes of L. monocytogenes , both live and heat-killed cells. MAbs 22D10 and 24F6—in combination with species-specific MAb EM-7G1—should be useful candidates for use in an ELISA sandwich assays for detecting L. monocytogenes in RTE meat and poultry products.     

Myoglobin Analysis for Determination of Beef, Pork, Horse, Sheep, and Kangaroo Meat in Blended Cooked Products

This method allows the concurrent detection of several types of animal muscle meat in comminuted, cooked meat products. Meat proteins were dissolved in a solvent containing 8M urea and dithioerythritol and separated by electrofocusing on an ultra-thin polyacrylamide gel, containing urea. The proteins were then transferred to nitrocellulose by electroblotting and the blot was incubated with anti-human myoglobin serum, a linking antibody, peroxidase-antiperoxidase (PAP) immune complex, and enzyme-substrate. Detection of less than 10% pork, horse and sheep meat was possible in a beef-based meat product which had been heated to an internal temperature of 120°C for 5 min. The method is not suitable for detection of chicken and turkey meats.