Application of a real-time PCR assay for the detection of ostrich (Struthio camelus) mislabelling in meat products from the retail market

A rapid and highly species-specific real-time polymerase chain reaction (PCR) assay has been developed for the authentication of ostrich meat (Struthio camelus). The method combines the use of ostrich-specific primers, that amplify a 155 bp fragment of the mitochondrial 12S rRNA gene, and a positive control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. SYBR^® Green dye or TaqMan^® fluorogenic probes were used to monitor the amplification of the target genes. Results obtained with the use of TaqMan^® probes as detection platform increased the specificity of the real-time PCR assay in comparison with the results obtained using SYBR^® Green. Analysis of 100 commercial ostrich meat products from the market demonstrated the suitability of the technique for the detection of ostrich DNA. The results obtained suggest that this method may be routinely applied to verify the correct labelling of ostrich meat products.     

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.     

Real-time PCR assay for detection of Trichinella in meat

Trichinella are a group of widely distributed parasites which have acquired high social relevance due to their involvement in foodborne infections caused by consumption of raw or undercooked food. A TaqMan^®-LNA probe Real-time PCR assay targeting the 5S rRNA was developed allowing the simultaneous detection of the 10 species and 3 genotypes of Trichinella present in meat tissues. The detection limit employing dilutions of genomic DNA was 2 pg and the determination of the detection limt in terms of ppm was 1 ppm.The main novelty of this work lies in the fact that it can assure the absence of 10 species and 3 genotypes of Trichinella in an only assay. The proposed methodology is rapid, robust, highly sensitive and readily adaptable in routine molecular diagnostic laboratories, and can be employed as molecular screening method in order to assess the food security.     

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.     

Survey of undeclared allergenic pistachio (Pistacia vera) in commercial foods by hydrolysis probe real-time PCR

Tree nut allergies represent an important health problem in industrialized countries. Among these, pistachio (Pistacia vera) kernels which are consumed as snack foods and used as ingredients in confectionery, chocolates, meat products, and ice-cream industries have been reported to cause IgE-mediated allergic reactions. Trace amounts of undeclared pistachio allergens can cause serious health risks for food-allergic consumers. In order to provide an appropriate method for the detection of pistachio in food products, a real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of pistachio was developed. The sensitivity was investigated on spiked wheat flour samples with defined raw and heat-treated pistachio contents (0.1–100,000 mg kg⁻¹). The real-time PCR detected pistachio in these mixtures down to the lowest investigated spike level of 0.1 mg kg⁻¹. In addition, analysis of different retail samples from the market was performed to demonstrate the suitability of the assay in the food industry. The real-time PCR results obtained from the analysis of 229 commercial food products revealed 29 that didn't declare pistachio or traces on the label but were found to contain pistachio. The presented real time PCR method is useful for relatively fast, highly selective, and sensitive detection of pistachio in food samples.     

Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening infections in neonates. The organism has been isolated from a wide variety of foods and environments. In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Cronobacter spp. in foods. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and 67 bacterial strains, including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully identified, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the assay in pure culture and formula infant without enrichment was 1.2 × 10³ CFU/ml (1.2 × 10¹ CFU/assay). After 24 h enrichment in broth, as few as 10⁰ CFU/ml or g of Cronobacter could be detected in artificially contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of 10⁸ CFU/ml Salmonella typhimurium in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of which two pork samples were found as positive by real-time PCR, whereas only one was detected by the ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp. in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.     

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.     

Prevalence,identification and molecular characterization of Cronobacter sakazakii isolated from retail meat products

Cronobacter sakazakii is an opportunistic foodborne pathogen predominantly in neonates with reported incidences in adults especially immunocompromised and the elderly. The current study was carried out to determine the prevalence of these organisms on meat products sold at Mansoura city, Egypt; besides its ability to compete with E. coli O157:H7 and Salmonella on their media. Suspected colonies were phenotypically-identified using API 20E system, additional biochemical tests, besides production of yellow-pigmented colonies; and genotypically-identified using species-specific PCR assays to detect the gene responsible for α-glucosidase activity, 16S rRNA gene and internal transcriber spacer sequence between 16S and 23S rRNA. Out of the 93 tested isolates, 14 isolates were confirmed as C. sakazakii with contamination rates of 16% (8/50) and 15% (6/40) of ground beef and beef burger samples, respectively and overall contamination rate of 15.6% (14/90) of all tested samples. The isolates were distributed among 7 different biogroups. These results added new epidemiological evidence about the widespread occurrence of these pathogens on tested meat products and such occurrence is an indicator for potential contamination with pathogens and so a health risk for consumers. Furthermore, C. sakazakii is an important competitor to E. coli O157:H7 and Salmonella on their selective media.     

Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR

We developed a multiplex real-time (RTi) PCR method for the simultaneous detection of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus using zot, vmrA, and vuuA as the respective target genes. A set of primer pairs specific for those target genes was designed and employed in the SYBR Green-based multiplex RTi-PCR assay. Quantitative analyses with ten-fold serially diluted genomic DNA of each target organism resulted in a linear correlation between C_T values and the amount of each target genome per reaction, with a lower detection level of less than ten genome copies per reaction. Similar sensitivities were observed for Vibrio-spiked seafood samples (oyster, crab meat, and raw fish). After 8 h of enrichment culture of the seafood homogenate in alkaline peptone water, our optimized multiplex RTi-PCR was shown to achieve theoretical maximum sensitivity (ca. 10⁰ CFU/gram food homogenate). Our proposed method is simple, robust and readily adaptable in routine laboratories, allowing for high-throughput surveillance of pathogenic Vibrio species in seafood.     

Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures

Natural antimicrobials are being more and more considered as alternative approach for controlling growth of microorganisms in food. The objective of this study was to evaluate the pomegranate extract’s (PE) potential to be used as a natural preservative in ready to eat meats. Listeria monocytogenes was the main target. In a preliminary assessment with the disk diffusion method PE showed inhibitory effect against all five tested species, in the following order of increasing sensitivity: L. monocytogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli and Staphylococcus aureus.No viable cells of L. monocytogenes were detected after incubation in BHI broth in presence of 7.5% v/v of the liquid PE (or 24.7 mg dry PE/ml). This concentration was considered as the Minimal Bactericidal Concentration (MBC) of the tested PE. Two pure components commonly found in PE, namely gallic and ellagic acids were also tested in BHI broth, however they did not show considerable inhibition of L. monocytogenes. PE in a concentration equal to the measured MBC was tested against L. monocytogenes in meat paté at different temperatures. At 4 °C during 46 days the extract inhibited the growth in meat paté by 4.1 log CFU/g compared to the control, which had reached log 9.2 CFU/g already on the 18th day. Inhibition was less pronounced at higher temperatures. The results indicate that the PE has a potential to be used as a natural preservative in meat products.     

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.     

Sextuplex PCR combined with immunomagnetic separation and PMA treatment for rapid detection and specific identification of viable Salmonella spp., Salmonella enterica serovars Paratyphi B,Salmonella Typhimurium,and Salmonella Enteritidis in raw meat

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 10¹ CFU/mL in pure culture and 10² CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.     

A PCR assay for sex determination of yak (Bos grunniens) meat by amplification of the male-specific SRY gene

Yak meat is of great economic importance to the people living in the cold high altitude area. In the present work, a multiplex PCR-based method was proposed for the sexing of yak meat by amplifying the target sequence of male-specific SRY gene. In a single reaction set, two DNA fragments of 121- and 290-bp were amplified from male meat DNA using two pairs of primers, but only one DNA fragment of 290-bp was amplified from female meat DNA. The assay proposed herein was applied to raw and heat-treated yak meat samples, and the identification results obtained were in perfect agreement with the anatomical sex of the yak meat samples. The method is fast, reliable and cheap. It is potentially suitable for the sexing of yak meat in routine analyses. It can be considered a helpful tool in the quality control of yak meat and meat products.     

Development of an efficient real-time PCR assay to quantify enterotoxin-producing staphylococci in meat products

A SYBR Green-based real-time PCR (qPCR) procedure for the rapid and specific detection of enterotoxin-producing Staphylococcus spp. in meat products has been developed. For this, a specific primer pair based on conserved regions of enterotoxin genes was designed for detecting most of the described staphylococcal enterotoxins. No cross-reactivity with other microorganisms or non-enterotoxin-producing Staphylococcus spp. was detected. The detection limits of the assay were about 2–40 cfu/g for artificially contaminated meat products after 8 h enrichment period at 30 °C. Total time for assay completion was approximately 12 h. Thus, the qPCR method offers a useful, rapid and efficient tool for screening enterotoxin-producing Staphylococci in meat products. This tool could be also used for monitoring these foodborne pathogens in food safety preventive programs.     

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.     

A sensitive HPLC-MS/MS screening method for the simultaneous detection of lupine,pea, and soy proteins in meat products

The use of vegetable proteins in various types of meat products is common practice. In order to control food specifications, also with regard to food fraud and allergenic potential, a reliable detection of these additives is required. Here, a sensitive screening method for the simultaneous detection of lupine (Lupinus angustifolius), pea (Pisum sativum), and soy (Glycine maxima) in meat products applying High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) has been developed. After protein extraction and tryptic digestion, 3 to 4 marker peptides for each plant species were measured by HPLC-MS/MS. For matrix calibration, emulsion-type sausages with 0, 1, 6, 32, 160, 800, and 4000 mg/kg raw legume protein isolates/legume flour were produced. The mentioned legumes were detectable in sausages with concentrations of 6 mg/kg legume protein isolates/legume flour or greater. High correlation coefficients (R² > 0.999) between the peak areas of the mass transitions of the marker peptides and the contents of legume proteins in the meat products were obtained. The limits of detection (LODs) of the method were about 5 mg/kg meat product for pea protein, 4 mg/kg meat product for soy protein, and 2 mg/kg meat product for lupine protein. No false-positive or false-negative results were recorded. The applicability of the described method was tested by analyzing commercial meat products with and without added legume proteins.     

A validated strip-based lateral flow assay for the confirmation of sheep-specific PCR products for the authentication of meat

Molecular methods, such as PCR and real-time PCR, have been developed to detect species in meat and meat products. Despite good specificity and sensitivity, they are not widely implemented in food control programs due to complex operation or financial reasons. In the present study, a simple, rapid and affordable method, Sheep-PCR-Strip [Sheep specific polymerase chain reaction-Strip], was developed for the authentic identification of raw and heat-treated mutton. The assay is based on PCR amplification of sheep DNA, followed by detection of the PCR product by a strip format; the result can be read within 5 min by the naked eye. There is a real advantage of the strip approach rather in the reduced time (5 min versus electrophoresis) and avoidance of chemicals (e.g. ethidiumbromide). The sensitivity of the Sheep-PCR-Strip test was established to be 0.01% for the detection of adulterated meat; the limit of detection (LOD) was up to 0.01 pg of sheep DNA. The assay was also specific for sheep, and no cross-reactions were observed in other non-target species. It is a promising new tool for sheep identification and can be rapidly modified for other meat detection and widely used for solving problems related to food quality assurance, species authentication and traceability.     

Transmission of Listeria monocytogenes from raw chicken meat to cooked chicken meat through cutting boards

Listeria monocytogenes (L. monocytogenes) is a food-borne pathogen contaminating poultry products. Ready-to-eat (RTE) cooked chicken meat can easily be contaminated with L. monocytogenes in post-processing activities. This study aimed to determine transmission of L. monocytogenes from raw chicken meat to hot and cooled chicken meat through polyethylene and wooden cutting boards. Raw chicken breast samples were purchased from retail markets and were artificially contaminated with L. monocytogenes at concentration of 7.35 ± 0.22 log CFU/ml. Contaminated raw samples were placed on polyethylene and wooden cutting boards to simulate bacterial transfer to cutting boards. Cooked chicken samples (hot and cooled) were then placed on the same cutting boards to simulate transfer of bacteria from cutting boards to cooked meat. L. monocytogenes successfully attached to polyethylene and wooden cutting boards and recovered after holding time up to 1 h. Transmissions of L. monocytogenes to cooled cooked samples from both types of cutting boards were relatively higher than hot cooked samples. Moreover, transfer rates of L. monocytogenes from wooden cutting boards at holding time of 1 h to both cooled and hot cooked samples were lower than those from polyethylene cutting board. It is recommended to use different cutting boards for raw and cooked materials and apply detergents and hot water for cleaning procedure to eliminate L. monocytogenes attached to the cutting boards and prevent cross-contamination of final products.     

Identification of meat species in pet foods using a real-time polymerase chain reaction (PCR) assay

Product mislabeling, adulteration, and substitution are increasing concerns in highly processed foods, including pet foods. Although regulations exist for pet foods, there is currently a lack of information on the prevalence of pet food mislabeling. The objective of this study was to perform a market survey of pet foods and pet treats marketed for domestic canines and felines to identify meat species present as well as any instances of mislabeling. Fifty-two commercial products were collected from online and retail sources. DNA was extracted from each product in duplicate and tested for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) using real-time polymerase chain reaction (PCR) with SYBR Green and species-specific primers. Of the 52 tested products, 31 were labeled correctly, 20 were potentially mislabeled, and 1 contained a non-specific meat ingredient that could not be verified. Chicken was the most common meat species found in the pet food products (n = 51), and none of the products tested positive for horsemeat. In three cases of potential mislabeling, one or two meat species were substituted for other meat species, but major trends were not observed. While these results suggest the occurrence of pet food mislabeling, further studies are needed to determine the extent of mislabeling and identify points in the production chain where mislabeling occurs.