Comparison of real-time PCR and ELISA-based methods for the detection of beef and pork in processed meat products

Two commonly used methodologies for species detection within processed meat products are real-time polymerase chain reaction (PCR), a DNA-based method, and enzyme-linked immunosorbent assay (ELISA), a protein-based method. In this study, a real-time PCR assay was compared to a commercial ELISA kit based on sensitivity, specificity, agreement among duplicate samples, cost, time, and ease of use. Fifteen reference samples containing known percentages (0.1–99.9%, w/w) of pork and beef were analyzed in duplicate using both methods. Thirty commercial products, including sausages, pet treats, and canned meats, were also tested in duplicate with each method. Reference sample analysis showed real-time PCR was able to detect pork in duplicate samples at 0.10% and beef at 0.50% in the binary mixtures. ELISA detected pork in duplicate samples at 10.0% and beef at 1.00% in the binary mixtures. When the results of reference and commercial samples were combined, real-time PCR demonstrated the greatest agreement among duplicate samples, at 96.7%, compared to 95.6% agreement for ELISA. The real-time PCR assay used in this study was found to be less expensive, while ELISA was less time-consuming and easier to perform. Both methods were successful at identifying species in ground meats, sausage, and deli meat samples; however, pet treats and canned meats proved more challenging. Overall, it was determined that the real-time PCR assay was optimal for species identification in processed meat products when a low detection limit is required; however, the ELISA kit may be advantageous in other situations due to its ease of use.     

Beef specific polymerase chain reaction assay for authentication of meat and meat products

The aim of this study was to develop species-specific polymerase chain reaction (PCR) assay for specific detection of beef using self-designed primer pair based on D-loop region of mitochondrial gene for amplification of 513 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The beef-specific primer pair was self-designed based on the available gene sequences on NCBI nucleotide database. The primer pair was individually optimized for amplification of desired 513 bp DNA fragments from isolated DNA of fresh beef. After successful amplification of desired DNA fragments by this primer pair, the PCR assay was evaluated for their efficiency to amplify DNA extracted from cooked and autoclaved meat and meat emulsion. The level of detection of this beef-specific primer pair was found to be less than 1 percent using PCR assay, even in admixed meat products containing meat of beef, buffalo meat, pork, chevon, mutton and chicken. No adverse effect of heat treatment, processing conditions and ingredients was observed on amplification pattern. The experiments were repeated for several time and results was found to be repeatable every-time.     

Identification of species in ground meat products sold on the U.S. commercial market using DNA-based methods

The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.     

A multiplex real-time PCR method for the quantitative determination of equine (horse) fractions in meat products

The recent horse meat scandal that rocked Europe in early 2013 shows how important it is for the routine food control authorities to constantly evolve analytical tools for the accurate evaluation of meat products among others, to ensure that product declaration and actual composition correlate. While in most cases qualitative detection approaches suffice for product evaluation, in other cases a quantitative analysis is important to distinguish between inadvertent contamination and deliberate adulteration.In this work an optimized real-time qPCR-based approach is presented and compared with another real-time based method for the detection of equine sequences among others in meat products. The method is a multiplex system for the simultaneous quantification of horse, beef, pork and sheep fractions, and was validated for use in the routine analysis of meat products. We describe a modular multiplex approach, where a quadruplex system (without sheep) and a pentaplex assay (with an integrated sheep detection system) can be applied in meat detection and quantification strategies. The analysis is matrix independent and relies on concomitant quantification of the animal species present in the food sample against the backdrop of myostatin, a universal sequence present in most mammalian and poultry species. The limit of detection of the analytical method was 10 genome copy equivalents. For validation of the method, meat samples comprising differing meat compositions were analysed, and the assay performed well in terms of robustness and reproducibility.     

Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures

Five species-specific Real-Time PCR protocols were developed, standardised and validated for the identification of turkey, chicken, beef, pork and sheep meat in complex food products. Specific primers and probes were designed for each assay. 16S rRNA and cyt-b target genes located in the mitochondrial DNA were used for this purpose. The limit of detection of the Real-Time PCR methods ranged between 0.02 pg and 0.80 pg of template DNA, with an efficiency between 95% and 100%. All methods were able to detect the target species when spiked at 1% in any other species and no relevant difference was observed between the Ct values of raw and cooked samples. An Internal Amplification Control was used to detect possible false negatives due to inhibitory substances eventually present in the sample matrix. The assays were tested on meat mixtures to evaluate the diagnostic sensitivity and specificity.     

Identification and verification of porcine DNA in commercial gelatin and gelatin containing processed foods

Gelatin, derived from bovine and porcine sources, has been used in many foods and pharmaceutical products. To ensure the compliance of food products with halal regulations, the reliable analytical methods are very much required. In this study, polymerase chain reaction (PCR) assay using species-specific primers was performed to evaluate the halal authenticity of commercial pure gelatin and gelatin-containing processed food products. Based on the specificity and cross-reactivity results of the seven species-specific primers by conventional PCR, the porcine species primer No. 2 was selected and it was able to detect species DNA in 12 out of 36 processed foods. The cloning, sequencing, and blasting at NCBI confirmed the presence of pork DNA in 5 out of 12 porcine DNA positive food samples. The maximum identity (homology) with pork sequence available in NCBI Gene Bank for the five samples ranged from 87% to 97% and the Query Cover ranged from 94% to 100%. The real-time PCR assay detected more positive samples (27 positive amplifications) compared to 12 positive samples with conventional PCR using porcine specific primer No. 2. PCR using species specific primers is a very useful and effective technique for halal authenticity of gelatin and gelatin-containing food products.     

Mislabelling of beef and poultry products sold in Malaysia

Meat species specification is important for consumer protection and increases concern in food labelling regulations enforcement. Although regulations exist for processed meat products, information on the prevalence of meat products mislabelling and regulatory compliance in Malaysia is lacking. In this study, 143 prepacked (beef and poultry) meat products (sausages, cold cut meats, cooked whole muscle meats, breaded products, meatballs and ground meats) were purchased from several national and international supermarket chains in Malaysia. These samples were analysed for the presence of common meat species (buffalo, cattle, chicken, goat, sheep, duck and goose) and meats prohibited by Islamic laws (“Haram”) (cat, dog, monkey, pig and rat) using species-specific primers. The results showed that 112 (78.3%) samples were mislabelled, attributed by the false declaration of species and/or presence of undeclared meat species. The mislabelled products consisted of 17/28, 3/4, 6/8, 19/25, 48/56, and 19/22 of sausage, cold cuts, cooked whole muscle meat, breaded product, ground meat, and meatball samples, respectively. Buffalo DNA was detected in 40 out of the 58 samples labelled as beef. The presence of undeclared chicken and buffalo DNA were detected in 33/58 and 62/84 of beef and chicken products, respectively. The five “Haram” meat sources, however, were not detected in all meat products tested. The presence of chicken or buffalo DNA in these products could be attributed to unintentional cross contamination from food processing equipment, especially meat grinder, and lack of proper cleaning or inadequate hygiene. In conclusion, this study shows that majority of the samples are not legally compliant, signifying that substitution and mislabelling of meat products are commonplace in Malaysia. Strict implementation of the Malaysia Food Regulations 1985 alongside with regular surveillance and monitoring programmes are compulsory to alleviate and deter mislabelling issues.     

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products.     

Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods

In the last decades, animal species identification became more important to prevent food adulteration. Here, we demonstrate the identification of seven poultry species, chicken, guinea fowl, pheasant, turkey, goose, duck and muscovy duck, through the use of the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods. DNA were isolated from poultry meat and meat products and were amplified with universal primers, designed for the mitochondrial 12S rRNA. Species-specific patterns and the reliable detection limit were identified as 0.5% for PCR and CE applications. Analyses of commercially available poultry products revealed fraud, as 6 of 36 contained undeclared species. The above-mentioned techniques are sensitive, reproducible and reliable for poultry species identification from foodstuffs.     

Polycyclic aromatic hydrocarbons (PAH) in Danish barbecued meat

Barbecuing is known to result in the formation of polycyclic aromatic hydrocarbons (PAHs). A validated method that employed pressurized liquid extraction (PLE), gel permeation chromatography (GPC) followed by solid phase extraction (SPE) on Silica and analytical determination by GC-MS was applied for the detection of 24 PAHs in barbecued meat. In total, 203 commercially barbecued meat samples (beef, pork, chicken, salmon and lamb) and 15 samples barbecued during controlled time and heat conditions were included. The sum of PAH4 (benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[b]fluoranthene) was highest for a pork tenderloin (195 μg/kg) and lowest for chicken breast (0.1 μg/kg) and controlled barbecued meat (<0.1 μg/kg). A worst case scenario, assuming daily consumption of barbecued meat, revealed a margin of exposure (MOE) between 7080 and 8450 for PAH4, lowest for commercial products. Even though this is less than 10,000, which according to the European Food Safety Authority (EFSA) would indicate a health concern for consumers, no consumer is believed to be exposed to these levels.     

Identification of duck,partridge, pheasant,quail, chicken and turkey meats by species-specific PCR assays to assess the authenticity of traditional game meat Alheira sausages

Game meat Alheira (Alheira de caça) sausage is a traditional fermented product typical from the Northeast region of Portugal, having bread and meats (including game) as main ingredients. It is a particularly appreciated product by consumers that commands higher prices, especially in comparison with the common Alheira produced with pork and poultry meats. Following our previous work in which several mammalian game meat species were successfully identified in game meat Alheira sausages for authentication purposes, the present work aimed at identifying game bird's species for the overall assessment of labelling compliance. For that purpose, several species-specific PCR assays targeting mitochondrial DNA for the detection of game and domestic bird's meat, namely duck, partridge, pheasant, quail, chicken and turkey were developed, optimised and applied to commercial samples of game meat Alheira for their authentication. The assays revealed a high specificity and sensitivity to detect the addition of all evaluated species down to a level of 0.01% (w/w). PCR results indicated the existence of several inconsistencies with the labelled information, namely the absence of declared game species (duck, partridge and pheasant) and the presence of undeclared poultry meat, pointing out to adulterations owing to substitution of game by domestic meat species. Since this is considered a high-valued traditional product that should be valorised and protected, this work puts in evidence the need for inspection programs to enforce regulation.     

Targeting double genes in multiplex PCR for discriminating bovine,buffalo and porcine materials in food chain

Beef, buffalo and pork are the major meat of economic, religious and health concern. Current methods to authenticate these materials in food chain are based on mainly single gene targets which are susceptible to break down by food processing treatments. We, for the first time, described here a double gene targeting short-amplicon length multiplex polymerase chain reaction assay for discriminating bovine, buffalo and porcine materials in a single assay platform. The advantage of the assay is evidenced in terms of fidelity, cost and time since it is highly unlikely that two different targets would be missing even in a decomposed specimen. Detection of multiple targets in a single assay definitely saves analytical cost and time. Mitochondrial cytochrome b (cytb) and ND5 genes were targeted and six different targets (length: 90–146 bp), two for each of cow (120 and 106bp), buffalo (90 and 138bp) and pig (73 and 146bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The detection limit was 0.02 ng DNA under pure states and 0.1% meat in binary mixtures and meatball products. Screening of Malaysian meatball products revealed all beef products were buffalo positive in which 35% were totally replaced. In contrast, all pork products were found uncontaminated from beef and buffalo.     

Identification of meat and poultry species in food products using DNA barcoding

DNA barcoding is a promising method for the sequencing-based identification of meat and poultry species in food products. However, DNA degradation during processing may limit recovery of the full-length DNA barcode from these foods. The objective of this study was to investigate the ability of DNA barcoding to identify species in meat and poultry products and to compare the results of full-length barcoding (658 bp) and mini-barcoding (127 bp). Sixty meat and poultry products were collected for this study, including deli meats, ground meats, dried meats, and canned meats. Each sample underwent full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were queried against the Barcode of Life Database (BOLD) and GenBank for species identification. Overall, full-barcoding showed a higher sequencing success rate (68.3%) as compared to mini-barcoding (38.3%). Mini-barcoding out-performed full barcoding for the identification of canned products (23.8% vs. 19.0% success), as well as for turkey and duck products; however, the primer set performed poorly when tested against chicken, beef, and bison/buffalo. Overall, full barcoding was found to be a robust method for the detection of species in meat and poultry products, with the exception of canned products. Mini-barcoding shows high potential to be used for species identification in processed products; however, an improved primer set is needed for this application.     

A high incidence of species substitution and mislabelling detected in meat products sold in South Africa

Due to their high market value, meat products are often targets for species substitution and adulteration. DNA-based methods are recognized as the most appropriate means to detect such fraudulent practices, however, these have not been extensively employed for the authentication of meat products available in South Africa. The aim of this study was to utilize a variety of molecular techniques to evaluate the extent of meat product mislabelling prevailing on the local market. A total of 139 processed meat products (minced meats, burger patties, deli meats, sausages and dried meats) were collected from retail outlets and butcheries in South Africa. The enzyme-linked immunosorbent assay (ELISA) was employed for the detection of undeclared plant proteins (soya and gluten) in the samples. A commercial DNA-based LCD array was used to screen the samples for the presence of 14 animal species, the results of which were confirmed by species-specific polymerase chain reaction (PCR) and in some cases also DNA sequencing. The results revealed that 95 of 139 (68%) samples contained species which were not declared on the product labelling, with the incidence being highest in sausages, burger patties and deli meats. Soya and gluten were identified as undeclared plant proteins in a large number of samples (>28%), while pork (37%) and chicken (23%) were the most commonly detected animal species. Unconventional species such as donkey, goat and water buffalo were also discovered in a number of products. Overall, this study confirmed that the mislabelling of processed meats is commonplace in South Africa and not only violates food labelling regulations, but also poses economic, religious, ethical and health impacts.     

Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan real-time PCR assay

A species-specific real-time polymerase chain reaction (PCR) assay using TaqMan^® probes has been developed for the identification of meat and meat products from common pigeon (Columba livia), woodpigeon (Columba palumbus) and stock pigeon (Columba oenas). The method combines the use of species-specific primers and TaqMan^® probes that amplify small fragments (amplicons < 200 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. The PCR assay reported in this work could be useful in inspection programs to verify the correct labelling of raw and heat-treated pigeon meat products.     

Real-time PCR for quantitative meat species testing

A method for quantitative meat speciation is described which combines the use of real-time PCR with species specific and ‘universal’ primers to measure individual species content and total meat content respectively. A comparison of the cycle number at which universal and species specific PCR products are first detected, in combination with the use of reference standards of known species content, is used as the basis for determining the percentage of a given species in a mixed sample. Importantly, the use of universal primers allows differences in DNA quality between samples and reference standards to be taken into account, while the use of real-time PCR allows measurement at an early stage in the PCR process which is inherently more accurate than the end point analysis associated with gel-based systems. This paper describes the quantification of beef in mixed samples to illustrate the principle of this approach.     

Prevalence of Campylobacter in chicken and chicken by-products retailed in Sapporo area,Hokkaido, Japan

The present work was carried out to study the prevalence of Campylobacter in fresh chicken meat and chicken by-products on retail level in Sapporo, Japan. Out of the 170 samples of chicken meat (breasts and thighs) and chicken by products (wings, livers, gizzards and hearts), 110 (64.7%) were contaminated with Campylobacter. Among the different products, chicken wings showed the highest contamination incidence (77.1%) followed by chicken thighs (70%), while chicken gizzards and hearts showed the lowest contamination incidence (45% and 40%, respectively). Of the 341 Campylobacter isolates, 278 (81.5%) were identified as Campylobacter jejuni and 63 (18.5%) isolates were identified as C. coli. All of the 341 Campylobacter strains identified by the conventional culture methods were further confirmed by polymerase chain reaction (PCR), which indicated that almost all (99.4%) of the tested strains were also positive by PCR. Screening of 195 selected Campylobacter isolates for determining their antimicrobial resistance indicated that most of the tested strains (73.3%) were resistant to three or more of antimicrobials examined. The study concluded that high proportion of chicken meat and chicken by-products marketed in Sapporo area are contaminated by Campylobacter, most of which are antimicrobial-resistant strains, with a possible risk from such microorganism especially from consumption of undercooked or post-cooking contaminated chicken products.     

Polymerase chain reaction detection of Listeria monocytogenes using oligonucleotide primers targeting actA gene

A polymerase chain reaction (PCR) assay targeting the gene encoding actA was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated pork, water and milk. 827-bp PCR product was detected in all 51 L. monocytogenes strains belonging to four different sero-groups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected both in all other Listeria spp., including Listeria innocua, Listeria ivanovill, Listeria seelingeri, Listeria welshimeri, or Listeria grayi and in non-Listeria bacteria, indicating that the primer set we use was highly specific for L. monocytogenes. The detection limit of the PCR assay for pure cell cultures was 10⁵ cfu per ml pure cell culture. However, the assay could detect as few as 10¹ cfu of L. monocytogenes in 25 g of pork and 25 ml milk and water following 16 h of enrichment in Listeria Enrichment broth (LEB) at 30 °C. Only a large number of dead L. monocytogenes cells can cause false positivity, as determined using model pork, milk and water samples artificially contaminated with decimal dilutions of dead L. monocytogenes. The total assay time including enrichment was approximately 18 h. These results suggest that the PCR assay based on amplifying actA gene can used to rapidly detect L. monocytogenes on pork, milk, water and possibly other types of food products.     

Application of an optimized 18-h method involving one step culturing and single primer-based PCR assay for detection of Salmonella spp. in foods

A polymerase chain reaction (PCR) using 20-mer oligonucleotide single primer (named primer 3) randomly designed on the basis of Salmonella Typhimurium gatD gene encoding galactitol-1-phosphate dehydrogenase could produce the specific DNA product of approximate 770-bp in all 38 Salmonella strains used. No 770-bp DNA band was amplified from any DNA samples of 20 non-Salmonella bacteria. The DNA band was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the RAPD–PCR assay for detection of pure genomic DNA from S. Typhimurium ATCC 13311 and Salmonella Enteritidis DMST 15676 was as few as 0.01 ng. When simple boiling in TE buffer for 5 min was used for extraction both Salmonella spp. DNA, the detection limits were at least 230 and 320 cells, respectively. By using the RAPD–PCR assay following at least 14 h pre-enrichment in nutrient broth (NB), as few as 1 CFU of S. Typhimurium ATCC 13311 or S. Enteritidis DMST 15676 per 25 g of autoclaved chicken meat was detected. When the optimized 18-h method involving pre-enrichment in NB and DNA extraction by boiling protocol followed by RAPD–PCR using primer 3 was evaluated in comparison with the conventional method on 195 possibly naturally-contaminated food samples, 36 samples were found positive by both methods. In addition, the results of the developed RAPD–PCR-based assay proved to be identical to those by the conventional method. The optimized 18-h method was simple, rapid and sensitive, achieved the same detection limit as the conventional method and produced a zero level of false-negative results.     

Production and preliminary characterization of monoclonal antibodies highly specific to pork fat protein

Meat and food products adulterated with pork fat concern Islamic consumers whose dietary laws prohibit consumption of pork meat. The objectives of this study are to investigate the existence of thermal stable-soluble protein (TSSP) in pork fat tissues and to develop and characterize monoclonal antibodies (MAbs) highly specific to the TSSP in pork fat tissues. The TSSP extracted was used as an immunogen. After cell fusion and cloning, seven monoclonal antibodies (MAbs; PF 2B8-3, 2B8-9, 2B8-20, 2B8-28, 2B8-31, 2B8-32, and 2B8-33) were developed and confirmed to be specific to pork fat protein without cross-reaction to other animal meats and fats in an indirect ELISA and Western blot. The ELISA assay based on MAb PF 2B8-31 can sensitively detect 1% pork fat protein mixed in other animal meats. These results support that the developed MAbs especially PF 2B8-31 can be sufficiently used as a bio-receptor in the development of immunoassay for the easy and rapid detection of pork fat adulteration. In addition, the ELISA based on the MAbs developed could be a useful tool to rapidly detect low levels of pork fat fraudfully adulterated in meat and food products.