Isabel González

PCR Identification of Beef, Sheep, Goat, and Pork in Raw and Heat-Treated Meat Mixtures

A PCR assay has been developed for the specific and qualitative detection of pork (Sus scrofa domesticus), beef (Bos taurus), sheep (Ovis aries), and goat (Capra hircus) in raw and heat-treated meat mixtures. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear species identification. Analysis of experimental meat mixtures demonstrated that the detection limit of the assay was 1% (wt/wt) for each species analyzed. This assay can be useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in meat mixtures.

Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures

A rapid real-time polymerase chain reaction (PCR) technique using SYBR Green detection system, has been developed for the quantification of red deer, fallow deer, and roe deer DNAs in meat mixtures. The method combines the use of cervid-specific primers that amplify a 134, 169, and 120bp of the 12S rRNA gene fragment of red deer, fallow deer and roe deer, respectively, and universal primers that amplify a 140bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The C(t) (threshold cycle) values obtained with the 18S rRNA primers are used to normalize those obtained from each of the cervid-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat treated binary mixtures of red deer, fallow deer or roe deer meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target cervid DNAs in the range 0.1-0.8%, depending on the species and treatment of the meat samples analyzed.

Differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes.

This work describes the differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR targeting sequences from two molecular markers: the mitochondrial displacement loop (D-loop) region and the nuclear melanocortin receptor 1 (MC1R) gene. A polymorphic D-loop fragment (∼270bp) was amplified and sequenced in a number of wild and domestic Sus scrofa meat samples, to find a nucleotide region suitable for PCR-RFLP analysis. Sequence data showed the presence of only a few point mutations across Sus scrofa D-loop sequences, not allowing direct discrimination between wild boar and domestic swine meats. Later, the MC1R gene was targeted and Sus scrofa-specific primers designed to amplify a 795bp MC1R fragment. Subsequent RFLP analysis of the MC1R swine-specific amplicons allowed selection of BspHI and BstUI endonucleases to carry out intraspecific Sus scrofa differentiation. Digestion of MC1R amplicons with the chosen enzymes generated characteristic PCR-RFLP profiles that allowed discrimination among meats from wild and domestic swine specimens. The technique also enabled the detection of samples that yielded heterozygous profiles, suggesting hybrids resulting from wild boar and domestic pig breeding. The PCR-RFLP reported here, targeting the MC1R gene may be routinely applied to verify the correct labelling of game products.

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs.

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.

Development of a combined PCR‐culture technique for the rapid detection of Arcobacter spp. in chicken meat

. A combined PCR‐culture technique was developed to detect Arcobacter spp. in fresh chicken meat. Following a short selective enrichment of chicken samples, bacterial DNA was extracted and amplified using primers targeted at the genes encoding 16S rRNA of Arcobacter spp. The selected primers amplify a 181‐bp fragment from all Arcobacter spp., whereas no PCR product is generated for other bacteria, including the closely related Campylobacter and Helicobacter species. The assay was used to screen 96 retail‐purchased chicken samples for the presence of Arcobacter spp. Fifty‐three percent of the samples analysed were positive for this micro‐organism. The assay is simple and sensitive and reduces the amount of time required to positively detect Arcobacter spp. in poultry meat.

Identification of Goose, Mule Duck, Chicken, Turkey, and Swine in Foie Gras by Species-Specific Polymerase Chain Reaction

A specific Polymerase Chain Reaction (PCR) has been developed for the identification of goose (Anser anser), mule duck (Anas platyrhynchos x Cairina moschata), chicken (Gallus gallus), turkey (Meleagris gallopavo), and swine (Sus scrofa domesticus) in foie gras. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of goose, mule duck, chicken, turkey, and swine in foie gras. Analysis of experimental mixtures demonstrated that the detection limit of the assay was approximately 1% for each species analyzed. This genetic marker can be very useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in foie gras.

Polymerase chain reaction-restriction fragment length polymorphism analysis of a short fragment of the cytochrome b gene for identification of flatfish species

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the specific identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides). PCR amplification of the cytochrome b gene using a universal primer together with a primer specifically designed as a part of this study produced a 201-bp fragment in all species analyzed. Digestions of the PCR products with Sau3Al, BsmAl, Rsal, and Mn/l endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 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.

Species-specific PCR for the identification of ruminant species in feedstuffs.

A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA mitochondrial gene has been developed for the specific identification of bovine, ovine and caprine DNAs in feedstuffs. The primers designed generated specific fragments of 84, 121 and 122pb length for bovine, ovine and caprine species, respectively. The specificity of the primers designed was tested against 30 animal species including mammals, birds and fish, as well as eight plant species. Analysis of experimental feedstuffs demonstrated that 0.1% of raw and heated bovine, ovine or caprine tissues can be easily detected using the species-specific primers developed. The performance of this method is not affected by prolonged heat treatment, and consequently it could be very useful to verify the origin of the raw materials in products submitted to denaturing technologies, for which other methods cannot be applied.

Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene.

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.