Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese

A polymerase chain reaction, amplifying a fragment of the mitochondrial DNA D loop region was developed for species specific detection of cattle and buffalo milk. The method was simultaneously extended for detection of HTST pasteurized milk samples and cheeses of bovine and buffalo origin. A common forward primer was used with two different species specific reverse primers that resulted amplification of a 126 bp and 226 bp products for cattle and buffalo, respectively, in simplex as well as in multiplex polymerase chain reaction. The primers successfully amplified DNA extracted by conventional protocol from minimal amount of raw milk, heat treated milk and cheese of either bovine or buffalo origin. The primers showed a high degree of specificity. The sensitivity of the assay was excellent with detection level of 0.1 percent adulteration of cow and buffalo milk or cheese (0.15 ng buffalo and 0.04 ng cattle DNA). The assay represents a sensitive and simple method for identification of adulteration in milk and cheese.     

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.     

Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods

The aim of our work was to evaluate a new commercial test kit for the detection of Listeria monocytogenes by PCR, using different DNA extraction methods. Food samples (pork sausage and “mozzarella” cheese) were spiked with known concentrations of L. monocytogenes and culture-enriched for 24 h. DNA extracted using three commercial kits and two standard methods, was amplified in species-specific PCR employing a L. monocytogenes PCR Detection Kit (Diatheva). The PCR-based method proved to be a reliable means of detecting the pathogen in food samples independently from the extraction procedure used, even for a contamination cell number of 1 cfu/g before culture enrichment. The molecular assay, showing perfect agreement with standard microbiological tests and a considerably shortened analysis time, provides a sensitive and rapid alternative for applications in the testing of foods for microbiological contamination, and highlights the potential of PCR technology in routine food control.     

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.     

Development of a real time PCR assay for detection of allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitized individuals. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a TaqMan real-time PCR method for specific detection of peanut in commercial food products. The assay uses two genetic makers in the Arah2 (125 bp) and ITS1 (90 bp) gene respectively, and TaqMan probes. The nuclear 18S rRNA gene was employed as a positive amplification control. Results obtained on sensitivity of both Arah2 and ITS primers with mixtures spiked with different concentrations of the target showed detection levels of 10 and 0.1 ppm, respectively. The applicability of the real-time PCR protocol to detect the presence of peanut DNA in commercial food products was determined through analysis of 123 different commercial products with the ITS primers which aimed higher sensitivity than Arah2 primer pair. The performance of the real-time PCR proposed herein allows a highly sensitive detection of peanut DNA in all the different food products, which declared to contain peanut material as well as in others were the presence of peanut or its traces wasn't declared in the labeling.     

A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods

Meat adulteration has posed considerable risks to public health. In this study, we developed a novel real-time quantitative PCR method for the detection of some mammalian and poultry species that are used as meat products or meat adulterants. The method was based on the detection of the single-copy nuclear gene myostatin. The specificity, heterogeneity, and copy number of myostatin were evaluated. Additionally, we determined the sensitivity and precision of the method. The results revealed that myostatin had high specificity and low heterogeneity among different mammalian and poultry species. The limit of detection was 5 pg of animal genomic DNA or 0.001% meat ingredient, and the limit of quantification was 10 pg of animal genomic DNA or 0.01% meat ingredient. The quantification results of 12 blind samples showed that the biases between the measured and true values were <25%. Therefore, the developed quantitative real-time PCR method for mammalian and poultry species is suitable for identification and quantification of different meat ingredients as a reference gene.     

Validation of a Real-time PCR assay for fast and sensitive quantification of Brucella spp. in water buffalo milk

Water buffalo milk and derived dairy products, including mozzarella cheese, represent a possible source of Brucella contamination for consumers. Brucella is a severe pathogen for human health even at low concentrations. It is therefore fundamental to develop an assay that is faster and more sensitive than the traditional bacterial culturing method for the detection of the pathogen in the food matrix. We designed a Real-time PCR assay able to detect as low as 1 CFU/ml of Brucella spp. in water (80% probability) and 3 CFU/ml of Brucella spp. in buffalo milk (50% probability) in less than 3 h without any enrichment step. The assay was validated by calculating specificity, sensitivity, detection limit, precision, PCR efficiency, DNA extraction efficiency and food matrix inhibition. When this method was employed to detect and quantify Brucella spp. in 109 buffalo milk samples, the assay demonstrated a higher sensitivity in comparison to bacteriological analysis (27 positive samples and 2 positive samples, respectively).     

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.     

Quantitative detection of goats’ milk in sheep’s milk by real-time PCR

The need to support food-labeling legislation has provided a driving force for development of analytical techniques for the analysis of food ingredients. In this study, the development of a method for quantification of goats’ milk in sheep’s milk mixtures is described. The technique involves the use of a real time PCR technique, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA). The method combines the use of goat-specific primers that amplify a 171 bp fragment from goat DNA, and mammalian-specific primers amplifying a 119 bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan) that hybridizes in the “goat-specific” and also in the “mammalian” DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C_t) at which mammalian and goat-specific PCR products are first detected, in combination with the use of reference standards of known caprine content, allows the determination of the percentage of goats’ milk in a milk mixture. The assay was used to analyze raw and heat-treated milk binary mixtures (goat/sheep), enabling the quantification of goats’ milk in the range 0.6–10%. The reported PCR assay may represent a rapid and straightforward tool applicable to the authentication of milk and other dairy products.     

Multiplex real-time PCR assays for simultaneous detection of maize MON810 and GA21 in food samples

This work describes a quantitative multiplex real-time PCR method optimized for the detection of maize MON810 and GA21. The use of specific primers and of labeled probes by real-time PCR allowed for the simultaneous detection and confirmation of amplicon identity and increased the reliability of the technique and the number of PCR applications to food analysis.Two different endogenous genes, Zein and Adh1, were evaluated for quantitative use as accountable for continuous development of maize traits. The quantification is based on a calibration standard curve obtained with the DNA extracted from Certified Reference Materials (CRMs). The limit of detection (LOD) and limit of quantification (LOQ) of the triplex assays developed was set at 3 and 36 copy numbers respectively.     

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.     

Applicability assessment of a real-time PCR assay for the specific detection of bovine,ovine and caprine material in feedstuffs

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of bovine, ovine and caprine processed animal protein (PAP) in industrial feedstuffs. The method uses species-specific primers and probes targeting short mitochondrial D-loop sequences, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 126 industrial feed samples that were manufactured to reproduce rendering processes of commercial feeds destined for farmed animals. The assay successfully classified samples as positive or negative according to the ruminant composition, enabling qualitative detection of banned material in feeds at levels as low as 0.1%. Although the method provides quantitative potential, results suggest that the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing treatments of the feeds, which affect the amount and quality of amplifiable DNA.     

Detection of verocytotoxin-producing Escherichia coli (VTEC) in minced beef and raw milk by colony blot hybridization

Verocytotoxin-producing Escherichia coli (VTEC) are foodborne pathogens that cause outbreaks linked to consumption of meat and raw milk. In this note the authors report results obtained from a survey conducted on minced beef and raw bovine milk samples using a Multiplex PCR (M-PCR) for the detection of eae, stx₁, stx₂ and hlyA genes as a screening step followed by a colony blot hybridization (CBH) technique for the isolation of the VTEC. Of 100 minced beef and 123 raw milk samples, 13 (13%) and 7 (5.7%) were positive in the M-PCR and among these 9 and 3 strains were isolated using CBH, respectively. All isolates showed the presence of the stx₂ gene, single or in association with the other investigated genes. None of the isolates belonged to the O157, O26, O91, O103, O111 and O145 serogroups. The study showed that the use of M-PCR for the screening of samples coupled with a sensitive and specific detection technique, could improve the possibility of detection of VTEC strains in foods. Moreover, the presence of VTEC in minced beef and in raw milk confirms their important role as putative vehicles of infection to humans. Stringent control of these foodstuffs is essential for food safety purposes.     

Event-specific qualitative and quantitative PCR detection of LY038 maize in mixed samples

With the development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative PCR detection methods have become the internationally agreed state-of-art. This paper describes the characterization and event-specific quantitative detection of LY038 maize insert with the application of reference molecule. The flanking regions were characterized by Inverse-PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right flanking sequence. In the qualitative PCR assay, two PCR systems were established with the event-specific and specie-specific primers respectively, and the limit of detection (LOD) was 0.1% (approximates to 37 haploid genome copies). In the quantitative TaqMan real-time PCR assay, a reference molecule was constructed by recombinant PCR and standard curves were set up. By using the reference molecule, we obtained standard curves with good linearity and relatively high efficiency of PCR reaction. The results indicated the usability of the plasmid as standard material. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for LY038 maize in this study were acceptable and suitable for LY038 maize detection in mixed samples.     

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.     

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.     

Prevalence,genetic diversity and antimicrobial susceptibility of Listeria monocytogenes isolated from open-air food markets in Greece

A total of 210 food samples originating from milk products, ready-to-eat salads, raw meat and raw meat products purchased in ten open-air market places in Thessaloniki, Greece, were analyzed for the presence of Listeria monocytogenes. Thirty (14.3%) contained L. monocytogenes with the highest prevalence in raw meat (27.5%), raw meat products (18%) and cheese (8%). The strains were susceptible to 16 antimicrobials as determined by microbroth dilution, except one strain which displayed resistance to tetracycline (MIC > 32 μg/ml). This strain carried the tetracycline resistance gene tet(M). Pulsed-field gel electrophoresis (PFGE) revealed a low genetic diversity among the isolates, irrespective of their origin. This suggests that dominant L. monocytogenes clones are widespread in different food product types in open-air food markets in Greece. The high prevalence of L. monocytogenes in these products indicates that appropriate hygienic measures and periodic bacteriological controls are also necessary in open-air food markets to reduce contamination with food-borne pathogens. Greek specialties made with raw meat and raw milk may contain L. monocytogenes and should not be consumed by persons at risk.     

A novel screening approach based on six real-time PCR systems for the detection of crustacean species in food

Analysis of crustacean species plays a role in authenticity issues as well as allergen detection. A real-time PCR-based screening assay was developed for the detection of crustaceans in food. In order to cover most relevant species in one analytical step, PCR systems were newly developed for the detection of shrimps (Penaeidae), lobster (Homarus sp.), Common shrimp (Crangon crangon), river prawns (Macrobrachium sp.) and Chinese mitten crab (Eriocheir sinensis). In addition a published system targeting Northern prawn (Pandalus borealis) was selected. All PCR-systems are based on mitochondrial 16S rRNA gene sequences and were optimized to be run with a standard program at a universal annealing temperature of 60 °C. Validation experiments confirmed a sensitivity of the PCR systems of 0.01–0.1 genome copies or 0.04–2.5 pg DNA, respectively. Specificity was demonstrated with 25,000 copies of pure genomic DNA from about thirty plant and animal species relevant in food and the performance in food matrix was evaluated. Finally primer and probes were pre-spotted steadily on 96-well PCR plates and the practical applicability of the assay was proven with selected food products. The assay enables detection of multiple species of market relevance.     

Development and evaluation of a real-time PCR assay for detection of lobster,a crustacean shellfish allergen

Crustacean shellfish are a leading cause of food allergy in American adults, and the Food Allergen Labeling and Consumer Protection Act requires that different types of crustacean shellfish be distinguished from each other. In general ELISA assays are not capable of differentiating crustacean type, but PCR assays are. In this work, a real-time PCR assay for lobster, a crustacean shellfish allergen, was developed and evaluated. Food matrices were spiked with lobster meat at 0.1, 1, 10, 100, 1000, 10⁴, and 10⁵ parts per million (ppm). In addition to testing of several different food matrices, method performance was determined using conditions which have historically proven challenging for PCR analyses, specifically food matrices with low DNA content and acidic pH levels, as well as foods that were treated with combined high temperature and pressure. Real-time PCR standard curves were generated from spiked, treated foods and analyzed with respect to linear range and reaction efficiency. In most cases, the assay was linear over 6–8 orders of magnitude; lower limits of detection were 0.1–1 ppm and reaction efficiencies were within the preferred range of 100 ± 10%. A notable exception occurred in the case of heat treatment at acidic pH, which resulted in severe delay or complete loss of amplification signals.     

Prevalence assessment of Coxiella burnetii and verocytotoxin-producing Escherichia coli in bovine raw milk through molecular identification

Raw milk has become increasingly appreciated by consumers and in the EU there is general acceptance of this product, the sale of which is regulated by EU Directives. However, unpasteurised milk can be contaminated by zoonotic agents, thus representing a risk for human health. The aim of our work was the assessment of Coxiella burnetii and verocytotoxin-producing Escherichia coli (VTEC) presence in bovine bulk tank milk of Marche region (Central Italy) destined to direct human consumption. PCR and real-time PCR well-established protocols have been used for pathogen identification, showing high sensitivity and specificity. C. burnetii prevalence was 27%, while VTEC serogroup were found as follows: 3.5% for both O157 and O145; 2.3% for O26; 4.7% for O103. In contrast, the immunomagnetic separation-based protocol (IMS) was able to identify only one sample (1.2%) contaminated by VTEC O157.These results indicate that a combination of real-time PCR and IMS proved to be more appropriate for VTEC serogroup identification, thanks to improved sensitivity. Moreover, the prevalence of these pathogens would suggest that non-O157 VTEC and C. burnetii should be included among microbiological criteria for raw milk, implementing control strategies to limit possible negative impact to consumers' health.