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.     

Development and application of molecular markers for poultry meat identification in food chain

Every year, large quantities of poultry and game meat are consumed. Thus, efficient techniques to identify the meat species origin are required which interest traders, consumers and organizations. In this study, two mitochondrial DNA (mtDNA) genes, Cytochrome b (Cyt b) and 12S ribosomal RNA (12S rRNA) were tested as putative discrimination markers in samples of raw and processed poultry meat (chicken, turkey, duck, goose, pheasant, partridge, woodcock, ostrich, quail and song thrush), applying the PCR–RFLP technique with universal primers and ten different restriction enzymes. Digestion of 12S rRNA by AciI successfully distinguished all avian species, producing species-specific patterns. We conclude that the 12S rRNA gene is more informative than Cyt b gene for avian species identification purposes. Moderate process treatment did not prevent the species identification, presenting similar patterns with the raw meat. Finally, this method was considered sufficient to detect mixtures of meat, making it a valuable tool for checking possible adulterations.     

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.     

Identification and antimicrobial resistance of Campylobacter species isolated from poultry meat in Khorasan province,Iran

Campylobacter is the main bacterial cause of acute gastroenteritis in humans. Campylobacter jejuni and Campylobacter coli are the most frequent Campylobacter species isolated from patients with diarrhea. Undercooked poultry meat is one of the main sources of human infection. Contamination of poultry carcasses by Campylobacter during processing occurs directly via intestinal contents or indirectly from bird to bird, via equipment and water. The objective of this study was to determine the prevalence and antimicrobial resistance patterns of Campylobacter spp. isolated from raw poultry meat in Mashhad, Iran. From May 2012 to July 2012, 300 poultry meat samples including chicken (150), turkey (70), partridge (50), and quail (30) were randomly purchased from retail outlets. Using cultural method and a PCR assay 49.7% of poultry meat samples were contaminated with Campylobacter spp. Campylobacter spp. were significantly (P < 0.05) more prevalent in chicken meat (59.3%), followed by partridge (48%), quail (40%), and turkey (34.3%) meat. The most prevalent Campylobacter spp. isolated was C. jejuni (80.8%); the remaining isolates were C. coli (19.3%). Overall, 96.6% Campylobacter isolates were resistant to one or more antimicrobial agent. Resistance to ciprofloxacin was the most common finding (81.9%), followed by resistance to nalidixic acid (73.2%) and tetracycline (67.8%). In conclusion, the results of this study showed the importance of chicken, quail, partridge, and turkey meat as potential sources of Campylobacter spp. infection in people.     

Antimicrobial resistance patterns of Campylobacter spp. isolated from raw chicken,turkey, quail,partridge, and ostrich meat in Iran

This study was conducted to determine the prevalence and antimicrobial resistance pattern of Campylobacter spp. isolated from retail raw poultry meats in Iran. From July 2009 to March 2010, a total of 494 raw meat samples from chicken (n = 200), turkey (n = 170), quail (n = 86), partridge (n = 17), and ostrich (n = 21) were purchased from randomly selected retail outlets in Shahrekord, Iran. Using cultural method, 187 meat samples (37.9%) were contaminated with Campylobacter. The highest prevalence of Campylobacter spp. was found in chicken meat (47.0%) followed by quail (43.0%), partridge (35.3%), turkey (28.8%), and ostrich (4.8%) meat. The most prevalent Campylobacter species was Campylobacter jejuni (92.0%). The PCR assay could identify 38 Campylobacter-contaminated samples that were negative using the cultural method. Antimicrobial susceptibility test results showed that 98.4% of isolates were resistant to one or more antimicrobial agents. Resistance to tetracycline was the most common findings (70.6%), followed by resistance to nalidixic acid (54.0%), and ciprofloxacin (49.7%). Significantly higher prevalence rates of Campylobacter spp. (P < 0.05) were found in meat samples taken in summer (51.1%). To our knowledge, the present study is the first report of the isolation of Campylobacter spp. from raw partridge meat in Iran.     

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.     

Using the PCR–RFLP method to identify the species of different processed products of billfish meats

A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method had been developed for the detection of five billfish species Xiphias gladius, Makaira nigricans, M. indica, Istiophorus platypterus and Tetrapturus audax in raw, frozen and heat-treated meats. The primers L-CYTBF and H-CYTBF were designed in the mitochondrial cytochrome b (cytb) gene and the molecular weight of amplified fragment was 348 bp and amplified the fragment from processed billfish meats. The results obtained from the BsaJI, Cac8I and HpaII enzymes digestion could be used to distinguish the five billfish species in frozen and heat-treated meats. Using the PCR–RFLP method, species of 10 commercial samples including raw fish fillets, frozen fish meats and fried fish meats could be identified. It was determined that two commercial samples of billfish products were not made from billfish. The method is sensitive, rapid and valid to detect fraudulent billfish products substituted from cheaper fish.     

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.     

PCR-based methodology for the authentication of grouper (Epinephelus marginatus) in commercial fish fillets

In an attempt to authenticate commercial frauds in fish fillets of grouper (Epinephelus marginatus) by being substituted with those of Nile perch (Lates niloticus) and wreck fish (Polyprion americanus), a polymerase chain reaction (PCR) method, based on the design of specific primers of these species was explored here. The oligonucleotides, which were designed from the 12S ribosomal RNA gene, generated PCR fragments of 100, 138 and 169 bp length for grouper, wreck fish and Nile perch respectively. The specificity of the primers was tested against more than 50 different fish species. Moreover, in this work 70 commercial fish fillets samples were analysed by this methodology; 58 out of them confirmed to be incorrectly labelled. The results suggest that this method may be a reliable tool for the detection of grouper adulteration. Also, this technique may help implementation of traceability systems in the seafood industry.     

Duplex real-time PCR assay for the simultaneous determination of the roe deer (Capreolus capreolus) and deer (sum of fallow deer,red deer and sika deer) content in game meat products

Due to the high price of game meat, food producers may be tempted to adulterate their products with cheaper meat. This paper presents a duplex real-time PCR assay which allows the simultaneous determination of the content of roe deer (Capreolus capreolus) and deer* (the sum of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon)) in food products to detect food adulteration. Relative quantification is carried out by using a reference (“all meat”) PCR assay based on the myostatin gene. The quantification approach was validated by analyzing binary meat mixtures with pork, “all game” meat mixtures containing each of the four game species in pork and a model game sausage. Compared to singleplex assays the duplex assay is time and cost saving. Thus, it is highly applicable to routine analysis in order to verify the authenticity of game meat products.     

A novel minisequencing test for species identification of salted and dried products derived from species belonging to Gadiformes

The aim of the present study is to develop an assay for the specific identification of Gadus morhua, Gadus macrocephalus, Gadus ogac, Molva molva and Brosme brosme targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 188 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the test were confirmed by fragment sequencing.     

Prevalence and antimicrobial resistance of Campylobacter species isolated from the avian eggs

Campylobacter is one of the most important food borne pathogens that cause bacterial gastroenteritis worldwide. The most commonly isolated species in humans are Campylobacter jejuni (C. jejuni) and Campylobacter coli. The emergence of antimicrobial resistance in Campylobacter spp. has been a growing public health concern globally. Information about antimicrobial resistance of Campylobacter at different levels of production is important for the development of control strategies for this pathogen. This study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter spp. isolated from different eggs from different avian species in Iran. A total of 440 egg samples were collected from different avian and analyzed for the presence of Campylobacter spp. in eggshell and eggs content under sterile conditions using Campylobacter selective agar base and the species were identified by biochemical tests. The suspected colonies were subjected to PCR assay for final confirmation as Campylobacter spp., and identification of C. jejuni or Campylobacter coli. Antimicrobial susceptibility testing was performed by the Kirby–Bauer disk diffusion method using Mueller Hinton agar. Campylobacters were detected in a total of 7 out of 100 (7%) eggshell of chicken samples and in 3 out of 60 (5%) eggshell of duck samples. In addition, Campylobacter spp. were also detected in 3.3%, 2.5%, 4.2%, 5% and 3.8 of the eggshell of goose, ostrich, partridge, quail and turkey samples, respectively. The overall prevalence rate of Campylobacter species from different avian eggs was found to be 7.7% (34/440). Among different avian egg samples, Campylobacter jejuni was more frequently isolated 28 (n = 28, 6.3%) than C. coli 6 (n = 6, 1.3%). In addition, the prevalence of C. jejuni was highest in summer and lowest in autumn. In this study Campylobacter spp. showed significant difference in resistance pattern with tetracycline and ciprofloxacin but gentamicin resistance was not found in both C. coli and C. jejuni isolates. Therefore, gentamicin is safe and effective drugs for the treatment of human campylobacteriosis if avian egg is considered as the source of infection.     

A new set of 16S rRNA universal primers for identification of animal species

In this study, bioinformatics were used to specifically design universal primers within 16S rRNA gene according to the following criteria: the priming sites needed to be sufficiently conserved to permit a reliable amplification (pooled samples) and the genetic marker needed to (a) be sufficiently variable to discriminate among most species and sufficiently conserved within than between species, (b) be short enough to allow also accurate amplification from processed samples (food) and non-invasive approaches (fur, feathers, faeces, etc.) (c) convey sufficient information to assign samples to species and (d) be amplified under variable lab conditions and protocols. Furthermore, short sequences allow the accurate massive inter- and intra-species identification of point mutations by the SSCP technique. The size of the amplified segment ranged from 222 to 252 bp. Amplification and identification success were 100% with all kinds of tissue tested in both raw and processed samples in a wind range of species, mammals (n = 27), fishes (n = 32) birds (n = 19), coleoptera (n = 23), reptiles (n = 5), crustaceans (n = 5) and cephalopods (n = 2), including almost all European mammal and avian game species. In addition, no intra-specific polymorphism was detected. Finally, gene fragments, homologous to those amplified by the primers used herein and retrieved from the GenBank for three animal sets [mammals (n = 248), birds (n = 231) and fishes (n = 644)] showed a particular precise percentage of correct identifications. Therefore, this short segment of the 16S rRNA mitochondrial gene could be a good candidate for a rapid, accurate, low-cost and easy-to-apply and interpret method to identify mammal and avian game species by PCR amplification and sequencing that can be easily incorporated in integrated conservation and forensic programmes.     

Genetic diversity of ivory shell (Babylonia areolata) in Taiwan and identification of species using DNA-based assays

For better traceability of seafood products in Taiwan, we need to effectively test product quality during the processes of identification of seafood species. The aim of this study was to analyse gene diversity and the methods of identification of high-value seafood, ivory shell (Babylonia areolata), on the Penghu Island in Taiwan using molecular marker technology and to build a relevant molecular database. Thirty-three samples of B. areolata and the samples of 5 other Babylonia species, including Babylonia feicheni, Babylonia spirata spirata, Babylonia perforata and Babylonia formosae formosae, from cultivation and from the wild were tested using the inter-simple sequence repeat (ISSR) method, mitochondrial DNA analysis, PCR-single-strand conformation polymorphism (PCR-SSCP) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that the primers ISSR3, ISSR7 and ISSR13 of the ISSR method and cytochrome C oxidase subunit 1 (coxI) gene analysis have a good discriminatory power in inter-species and intra-species tests. In conclusion, ISSR, PCR-DGGE and PCR-SSCP with coxI analysis can be used for the screening and identification of B. areolata species. Furthermore, these molecular methods could be useful for the identification of other types of seafood.     

A qualitative risk assessment of the microbiological risks to consumers from the production and consumption of uneviscerated and eviscerated small game birds in the UK

Since the beginning of the 21st century, consumption of wild game birds has been increasing, concurrent with a rise in consumer interest in free-range and ‘healthy-eating’ foodstuffs. Game birds can carry zoonotic pathogens, predominantly within the viscera. Whilst removal of the viscera is normal practice during the dressing of game birds, there is a specialised market in the UK for certain small game birds that are consumed uneviscerated. This qualitative risk assessment evaluates the risks to the consumer from the production and consumption of both uneviscerated and eviscerated small game birds for Salmonella spp., Escherichia coli (verotoxigenic), E. coli (antimicrobial resistant), Campylobacter spp., Toxoplasma gondii, Chlamydophila psittaci and Listeria monocytogenes. Whilst most pathogen/bird combinations were considered to have a very low risk, results suggest that Campylobacter spp. and T. gondii can pose an increased risk to consumers for some species of wild game birds. There was no greater risk associated with the consumption of uneviscerated game birds than for eviscerated birds. In some cases, the risk from eviscerated birds may even be slightly higher, as the risk of cross-contamination during evisceration can outweigh the reduction in pathogenic organisms due to removal of the viscera. Additionally, the current low frequency of consumption of uneviscerated game birds of most species reduced the overall risk estimate for these birds. If there is an increased frequency of consumption in the future, then this risk should be re-examined. Assuming a general level of compliance with regulations and basic hygiene practices, the results suggested that large outbreaks of infection among UK consumers are unlikely, but sporadic, infectious events could occur due to combinations of ‘rare-event, hygiene-related issues’ in the ‘field-to-fork’ chain and/or inadequate cooking of the bird.     

A multiplex-conventional PCR assay for bovine,ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific

A multiplex PCR assay was developed for rapid and reliable identification of bovine, ovine, caprine and fish species in feedstuffs simultaneously. The method merges the use of bovine, ovine, caprine and fish primers that amplify fragments (ovine; 119 bp, caprine; 142 bp, fish; 224 bp and bovine; 271 bp) of the mitochondrial t.glu gene forward and cyt b reverse, 12S rRNA, 12S rRNA, and ATPase subunit 8 genes respectively, and a universal 18S rRNA primers that amplifies a 99 bp from eukaryotic DNA. To evaluate the effect of heat treatment, a severe sterilization condition (133 °C at 300 kPa for 20 min) was applied. Multiplex analysis of the reference feedstuff samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this multiplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for bovine, ovine, caprine and fish species in feedstuffs.     

Rapid species identification of fresh and processed scallops by multiplex PCR

Food control policies regarding to seafood label authenticity have become a global issue due to increased incidence of species substitution or mislabelling. Proper species-level identification in processed scallop products is hindered by the lack of morphological characters such as their valves. In order to identify four commercially important scallop species (Argopecten purpuratus, Argopecten irradians, Mizuhopecten yessoensis, Pecten albicans) a species-specific multiplex PCR reaction is described herein. Novel reverse species-specific primers in combination with one universal forward primer designed to amplify a partial region of the mitochondrial 16S rRNA gene were assayed in fresh as well as in manufactured scallop samples. All PCR reactions showed a high specificity allowing an unambiguous species authentication.     

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.     

Development of a Real Time PCR system for detection of ochratoxin A-producing strains of the Aspergillus niger aggregate

Members of Aspergillus section Nigri are distributed worldwide, being considered as common food spoilage fungi. Some species of this section produce ochratoxin A (OTA), mainly Aspergillus carbonarius and several members of the Aspergillus niger aggregate. Detection of ochratoxigenic A. niger aggregate strains is important to prevent OTA contamination in foodstuffs. A new Real Time PCR procedure has been developed for the rapid and specific detection and quantification of ochratoxin A-producing strains of the A. niger aggregate. Two specific primers delimiting a 120 bp fragment and a probe were designed and directed to a polyketide synthase (PKS) from A. niger CBS 513.88 genome. This PKS has a strong similarity to PKS of A. ochraceus fragment involved in ochratoxin biosynthesis. Specificity was confirmed by testing primers towards purified DNA from 91 fungal strains, including reference and food isolates. The SYBR-Green and the TaqMan approaches developed allowed the specific detection only of ochratoxigenic strains of the A. niger aggregate. All other analyzed food related fungi gave negative results. This is the first report on a Real Time PCR system for the detection of OTA-producing strains of the A. niger aggregate.     

A multiplex PCR assay for fraud identification of deer products

Attempts were made to established one-step multiplex PCR assay for the fraud identification of the mostly used species in deer products (bovine, ovine, porcine and poultry). Primers were selected from published papers or designed in the well-conserved region of tRNA-Val and 16S rRNA mitochondrial genes after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 124, 183, 225 and 290 bp length for bovine, poultry, ovine and porcine, respectively. The detection limit was 1 ng for porcine and ovine primers, 5 ng for poultry primers and 0.5 ng for bovine primers. The results demonstrated that fraud phenomena are very epidemic in the deer products, especially heart, blood, penis and antler products.The multiplex PCR described in this study, proved to be very sensitive and reliable in species identification, could be considered as a further improvement of traditional methods based assay for the identification of deer products.