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.     

Virucidal Effectiveness of Flexible Pouch Processing of Meat Products Prepared from Foot-and-Mouth Disease-Affected Cattle

Thermal processing of ground beef, beef cubes or beef slices in commercially popular flexible pouches, effectively inactivated foot-and-mouth disease (FMD) virus that had resulted from infection of cattle. Cooking the meat products at 75°C for 20 min or 80°C for 15 min was virucidal; however, FMD virus survived in ground beef products processed for shorter time periods and/or at lower temperatures. The processing schema described were limited to 200g samples of these meat products. Postmortem decrease of pH due to electrical stimulation of carcasses inactivated the virus in skeletal muscle but not in lymph mode tissue.     

Procedure for Soluble Collagen in Thermally Processed Meat Products

Methods to determine the extent of collagen solubilization during thermal processing in a product such as frankfurters may not be accurate. We modified existing procedures to separate soluble collagen from thermally processed meat samples without solubilizing more collagen. Water bath temperatures (36, 40, 44, 48, 52 and 56°C) and times (5, 10 and 15 min) were studied in a complete factorial design. Soluble collagen increased up to 48°C and 10 min, leveling off beyond that. In a 48–52°C water bath 10–15 min was sufficient to separate soluble collagen from thermally processed meat products without solubilizing more.     

Storage studies on a sous vide spaghetti and meat sauce product

Shelf-life studies were done on a minimally processed sous vide spaghetti and meat sauce product subjected to a heat processing at 65°C and 75°C equivalent to a 5D and 13D reduction of Streptococcus faecium and subsequently stored at refrigeration (5°C) or under mild temperature abuse storage conditions (15°C). Products stored at 5°C had a shelf-life of ≥35 days irrespective of the processing treatment. However, for products stored at 15°C, packages were visibly swollen due to additional carbon dioxide production after 14 to 21 days, depending on the severity of the heat processing treatment, and the product had a distinct fruity odor when opened. Initial spoilage micro-organisms consisted predominantly of Bacillus species. However, after 7-12 days at 15°C, the spoilage pattern changed, with lactic acid bacteria accounting for almost all of the spoilage micro-organisms. As lactic acid bacteria increased in numbers, there was a concomitant increase in carbon dioxide and lactic acid production and a drop in pH of the product. This study has shown that physical, chemical and microbiological changes can be useful indicators to demonstrate temperature abuse of a minimally processed pasta product.     

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.     

Application of a polymerase chain reaction (PCR) method as a complementary tool to microscopic analysis for the detection of bones and other animal tissues in home‐made animal meals

A polymerase chain reaction (PCR) method was compared with a variation of the official microscopic technique (Directive 98/88/EC) for the detection in animal meals of cereals (wheat and corn) and animal parts (bone, feathers, meat, liver, fat and blood). Microscopy successfully detected animal bones in raw feeds with a sensitivity of 1 g kg^?1, while the sensitivity of the PCR method was in the range of 5–10 g kg^?1. Microscopy also allowed the detection of animal bones and feathers in feeds processed at 115 and 133 °C but failed to detect other animal materials. The PCR method successfully detected cereals (wheat and corn) as well as meat, bone, liver, fat and feathers after processing at 115 °C for 20 min. Heating at 133 °C under overpressure (autoclave) conditions resulted in more intense DNA fragmentation and lower DNA extractability. Nevertheless, bone and liver, as well as wheat and corn in home‐made animal meals, were successfully detected even after heating at 133 °C for 20 min. Copyright © 2004 Society of Chemical Industry     

Biochip Technology for the Detection of Animal Species in Meat Products

The verification of declared components in meat products is an essential task of food control agencies worldwide. To date, the ELISA and species-specific polymerase chain reaction (PCR) are two commonly applied analytical tools employed by many authorized food control laboratories. These trusted methods however do not allow the simultaneous detection of all the animal species present in a meat sample. Additionally, detection of undeclared components resulting from inadvertent contamination or deliberate adulteration of the meat products requires additional processing of the samples, resulting in increased expenditure. The use of DNA biochip analysis that allows simultaneous processing of many meat products, while concomitantly generating results for the detection of all animal species present in the meat products is thus highly desirable. In this work, two commercially available animal chip detection systems (CarnoCheck Test Kit and MEAT^species LCD Array) are compared in terms of sensitivity, robustness, reproducibility, and ease of handling. The two animal species differentiation biochip methods compared well in efficiency and could simultaneously detect from eight to 14 animal species in the meat products. Detection limits were found to be in the range of 0.1% to 0.5% in meat admixtures, with good reproducibility of results. More than 70 commercially available meat samples were analyzed in this work, with the results validated against traditional PCR methodology. Both biochip methods performed well and could be implemented for routine use in any food control agency.     

Electronic nose analysis of volatile compounds from poultry meat samples,fresh and after refrigerated storage

Electronic nose technology has previously been applied to the assessment of the quality of red meats, pork and fish, but not poultry products. In the present study the ability of the electronic nose to assess the microbiological quality of raw poultry meat as a function of storage time and temperature was investigated. Four types of chicken pieces (boneless breast with and without skin, wings and thighs) were stored for up to 2 days at 13 °C (the maximum allowable temperature in poultry processing environments) or for up to 5 days at 4 °C (refrigeration temperature for raw poultry products prior to shipping or further processing). Saline rinses of meat samples were serially diluted in tryptic soy broth to 10^?10. The rinses and their associated serial dilutions were analysed on an electronic nose with 12 metal oxide sensors in order to determine the specificity and sensitivity respectively of the assay. Principal component analysis (PCA) maps of the data confirmed that the electronic nose could differentiate volatile compounds associated with individual types of meat samples properly stored at 4 °C from those maintained at processing temperature, 13 °C, for a comparable time, even as early as day 1 of storage. Differences in headspace gases from any type of meat sample stored at one temperature could also be determined with increased storage time. However, data from samples stored at 4 °C clustered more tightly in PCA maps than those associated with samples maintained at 13 °C, indicating a greater diversity in volatile compounds at the higher temperature. We have shown herein that the electronic nose can detect changes in the volatile compounds associated with chicken meat based on product storage time and temperature; the technology can assess length of sample storage as well as deviation from refrigeration temperature. Published in 2002 for SCI by John Wiley & Sons, Ltd.     

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.     

Residual glutamic-oxaloacetic transaminase (GOT) activity in thermally processed poultry and poultry products as an indicator of end-point temperatures

Glutamic-oxaloacetic transaminase (GOT) activities in chicken and turkey thigh and breast meat samples, thermally processed at 60–84°C in a model heat-treating system, were evaluated for use as indicators of end-point cooking temperatures (EPT). Wings, breasts, thighs and legs from commercially cooked, whole, roasted chickens and commercially processed products containing chicken and turkey meat were analyzed also to determine if residual GOT activities would indicate compliance with recent FDA/FSIS EPT recommendations. Activities of samples processed in the model system decreased logarithmically with increasing temperatures. GOT activities were higher (P < 0·05) in thigh meat than breast meat in both chicken and turkey samples; activities were higher in turkey than chicken. GOT values for chicken thigh and breast meat at 74°C, the FDA/FSIS recommended EPT for use by food handlers and retailers, were 735 and 164 Sigma-Frankel units ml^?1 (SFU ml^?1), respectively. Values for turkey thigh and breast meat at this temperature were 1080 and 450 SFU ml^?1, respectively. The range of activities was 7–13 SFU ml^?1 in commercially prepared chicken products and 27–161 SFU ml^?1 in turkey products. Analysis of these products showed adequate cooking and compliance with FDA/FSIS recommended EPT for retail sale. These data indicate that residual GOT activity in processed poultry has potential for use as an indicator of EPT.     

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.     

Recombinant DNA in meat additives: Specific detection of Roundup Ready™ soybean by nested PCR

Soybean proteins are widely used by the meat industry as technological coadjutor when producing processed products such as emulsified and ground meat products. Since regulations for the use and labeling of GMOs and derived ingredients are in force in Brazil, a PCR‐based method capable of detecting Roundup Ready^? (RR) soybean was employed for meat additives. Thirty‐two samples of meat additives containing soy proteins were tested for the presence of soybean amplifiable DNA and RR soybean DNA. Twenty‐five samples gave a positive signal for the lectin gene, confirming the presence of soybean amplifiable DNA and 15 samples returned a positive signal for specific RR detection confirming the presence of genetically modified soy. These results demonstrate for the first time the presence of RR soybean in meat additives. This method may be useful for meat industries interested in controlling the presence of RR soybean in additives used for meat products manufacture. Copyright © 2007 Society of Chemical Industry     

Differentiation of toxigenic Staphylococcus aureusin staphylococcal isolates from prepared and frozen foods by combined arbitrarily primed polymerase chain reaction and DNA probe

In prepared and frozen flamenquín and hake fish fingers Staphylococcus aureus as sanitary hazards have been detected. In the present work, a combined method that includes an arbitrarily primed PCR (AP‐PCR) and a mixed DNA probe hybridisation designed for the enterotoxigenic genes sea, seb, sec, and sed will be assayed to differentiate enterotoxigenic S. aureus from other staphylococcal species isolated during the processing of prepared and frozen foods. From the protocols tested for the AP‐PCR, the highest number of amplification bands showing the best resolution was achieved at 30°C annealing and 35°C extension temperatures. Several staphylococci identified by a biochemical test as S. aureus showed in the AP‐PCR analysis different banding patterns to the references S. aureus. The isolates, were investigated by slot blot hybridisation for genes encoding A, B, C, and D staphylococcal enterotoxins to determine their enterotoxigenic potential. Several isolates characterised by the AP‐PCR analysis as S. aureus hybridised with the DNA probe mixture. The combined AP‐PCR and DNA probe hybridisation assayed was able to differentiate toxigenic S. aureus from other staphylococcal species from prepared and frozen foods. This method could be considered as microbial quality assurance in these products.     

Optimization of conditions for profiling bacterial populations in food by culture-independent methods

In this study we used culture-independent methods to profile bacterial populations in food products. Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) were employed in order to identify bacterial species without the need of isolation and biochemical identification. The protocols used to extract the DNA, subsequently subjected to PCR amplification for DGGE, as well as the hybridization procedure for FISH, were optimised. Moreover, an extensive study on the primers and probes to be used for the direct detection and identification of microorganisms commonly found in food, was carried out. Meat and cheese samples, fresh or processed, were subjected to DGGE and FISH analysis and the results obtained highlighted how the processing in food industry is decreasing the bacterial biodiversity. Not only processed cheese or meat but also fermented products were dominated by only one or few species. Lactobacillus sakei, Lactobacillus curvatus and Brochothrix thermosphacta were the main species found in meat products, while in cheese(s) Lactococcus lactis, Streptococcus thermophilus and Leuconostoc spp. were repeatedly detected. The results obtained by the two culture-independent methods used always correlated well.     

Enzyme Profile of Raw and Heat-Processed Beef, Pork and Turkey Using the "Apizym" System

A semi-quantitative micromethod enzyme system (APIZYM) designed for the detection of 19 individual enzymes was used to determine the presence and level of activity of these enzymes in raw and heat processed (60 and 71.1°C) beef, pork and turkey muscle tissue. Filtrates were obtained from 0.9% saline extracts of the raw and heat processed samples. Heat processing the product to 60°C greatly decreased the number of enzymes that could be detected. Samples heated to 71.1°C exhibited little enzymatic action, except for minimal activity of leucine aminopeptidase. Thus, indicating the possibility of using the APIZYM system as a new and/or improved method for determining the end-point temperature to which meat and poultry products have been heat processed.     

Determination of the toxic variability of lipophilic biotoxins in marine bivalve and gastropod tissues treated with an industrial canning process

Contamination of shellfish with lipophilic marine biotoxins (LMB), pectenotoxins (PTXs), yessotoxins (YTXs) and okadaic acid (OA) toxin groups in southern Chile is a constant challenge for the development of miticulture considering the high incidence of toxic episodes that tend to occur. This research is focused on using methodologies for assessing the decrease in toxins of natural resources in Chile with high value, without altering the organoleptic properties of the shellfish. The species were processed through steaming (1 min at 121°C) and subsequent canning (5 min at 121°C). Changes in the profiles of toxins and total toxicity levels of LMB in endemic bivalves and gastropods were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total reduction of toxicity (≈ 15%) was not related to the destruction of the toxin, but rather to the loss of LMB on removing the shells and packing media of canned products (***p < 0.001). Industrial processing of shellfish reduces LMB contents by up to 15% of the total initial contents, concomitant only with the interconversion of PTX-group toxins into PTX-2sa. In soft bottom-dwelling species with toxicities beyond the standard for safe human consumption (≥ 160 μg OA-eq kg^–1), toxicity can be reduced to safe levels through industrial preparation procedures.     

Determination of 14 aminoglycosides by LC-MS/MS using molecularly imprinted polymer solid phase extraction for clean-up

An LC-MS/MS method for screening 14 aminoglycosides in foodstuffs of animal origin is presented. Its scope includes raw materials and processed ingredients but also finished products composed of milk, meat, fish, egg or fat. Aminoglycosides are extracted in an acidic aqueous solution, which is first recovered after centrifugation, then diluted with a basic buffer and finally purified by molecularly imprinted polymer-solid phase extraction (MIP-SPE). Analytes are detected within 8 min by ion-pair reversed phase LC-MS/MS. Due to the large range of foodstuffs involved, the variability of matrix effects led to significant MS signal variations. This was circumvented by systematically extracting each sample twice, i.e. ‘unspiked’ and ‘spiked’ at the screening target concentration of 50 µg kg^?1. The method was validated according to the European Community Reference Laboratories Residues Guidelines giving false-negative and false-positive rates ≤3% for all compounds. Ruggedness of the method was further demonstrated in quality control operations by a second laboratory. The 14 aminoglycosides in water-based standard solutions were stable for up to 6 months when stored at either ?80°C, ?20°C or at 4°C storage temperatures.     

Species identification and animal authentication in meat products: a review

Meat species identification and animal authentication in meat products is a significant subject, attention to which would contribute to fair-trade, and would enable consumers to make informed choices. Analytical methods are often based on protein or DNA measurements. Methods based on protein fractions include electrophoretic, chromatographic and immunological techniques and are often not suitable for compound food products, nor are they sensitive in processed products to differentiate closely related meat species. Advances in DNA technology have led to the rapid development of alternative approaches to species identification. Recently, application of polymerase chain reaction in food analysis has increased in the light of their simplicity, specificity and sensitivity. This review discusses a wide range of analytical methods with a focus on their ability to quantify meat and authentication of meat products.