Detection of hazelnut (Corylus spp.) in processed foods using real-time PCR

Hazelnut seeds (Corylus spp.) are source of allergens and could cause severe adverse reactions in sensitized subjects, even if consumed in traces. This work presents two sensitive real-time PCR methods to quantify hazelnut in foods, one using the Sybr green dye and one based on hazelnut-specific Taqman probe designed on Cor a1.04 gene (specific amplicon: 82 bp). The sensibility and the robustness of the method were estimated analyzing spiked samples and some commercial hazelnut-containing creams. The lowest detection limit was 0.1 ng of genomic DNA. A qualitative specific PCR and a comparison of different DNA extraction protocols are also discussed.     

Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.     

Simultaneous detection of Listeria species isolated from meat processed foods using multiplex PCR

Listeriosis is a foodborne disease caused by the pathogenic Listeria monocytogenes and is considered as a serious health problem due to the severity of symptoms and its high mortality rate. Listeria genus is divided into six species and especially L. monocytogenes is an important foodborne pathogen in humans and livestock. Recently, other Listeria species are reported as pathogenic strains in decayed foods and environments as well. High mortality rate of listeriosis demands for rapid methods to detect the potential presence of the food pathogens in the food industry. We have developed a multiplex PCR for rapid and simultaneous detection of six Listeria species including Listeria grayi, Listeria innocua, Listeria ivanovii, L. monocytogenes, Listeria seeligeri and Listeria welshimeri to identify specific Listeria species in processed foods. The optimized multiplex PCR in this study utilized one Listeria genus specific and each Listeria species-specific primer pairs. Each primer pair yields the products of 370-bp for Listeria genus-specific, 201-bp for L. grayi-specific, 749-bp for L. innocua-specific, 463-bp for L. ivanovii-specific, 509-bp for L. monocytogenes-specific, 673-bp for L. seeligeri-specific and 281-bp for L. welshimeri-specific. We have successfully applied multiplex PCR strategy to 93 Listeria isolates from processed meat products to determine specific Listeria species and out of which 81 strains of L. monocytogenes, 10 strains of L. innocua and 2 strains of L. welshimeri were identified. This established multiplex PCR provides rapid and reliable results and will be useful for the detection of Listeria species in contaminated food products and clinical samples.     

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.     

Applicability of quantitative PCR to soy processed foods containing Roundup Ready Soy

Detection methods for genetically modified organisms (GMO) have been developed among countries, but there are still no practical quantitative methods for the processed foods. Although the quantitative methods in the Japanese standard are now targeted at raw grain, we examined whether the methods may also be applied to processed soy foods. The Roundup Ready Soy (RRS) in soy products by our own or professional making were quantified using real time PCR, and we compared the RRS content with that in raw soybeans. As a result, sufficient copy numbers of the lectin and introduced gene were obtained for quantification from all the soy products. Almost all items showed no significant differences in the RRS content. These findings suggested that the Japanese official quantitative method might be useful for screening in some soy products.     

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.     

Droplet digital PCR for routine analysis of genetically modified foods (GMO) – A comparison with real-time quantitative PCR

Droplet digital polymerase chain reaction (ddPCR) has seen increasing applications in recent times, also in the analysis of genetically modified (GM) food and feed samples. While quantitative real-time PCR (qPCR) methods have been traditional mainstays till now, the applicability of ddPCR in routine analysis of GM food and feed has not yet been widely demonstrated. In this work, we applied ddPCR on selected GM-food and feed samples that were recently analyzed on the qPCR platform in inter-laboratory proficiency tests and showed good performance of the ddPCR method. Sometimes GM DNA at different transgene levels, useful as reference material is not readily available. Applying ddPCR, different concentrations of GM material, specifically transgene DNA at different levels (0.1–10%) useful as reference DNA, were generated from 100% non GM material and 100% transgene plant material respectively, and key performance parameters of the ddPCR assay evaluated. DdPCR performed well, indicating its suitability for the production of reference GM materials. In an expanded analysis, DNA extracted from a 100% GM reference soy plant (CV127) was appropriately diluted to low copy numbers and the absolute LOD_95% determined at 2 copies (nominal value), comparing well with various published qPCR assays. In our inhibition studies, ddPCR showed a clear advantage over qPCR in SDS-inhibited samples, while its tolerance to other tested inhibitors was comparable with qPCR. Significantly, the qPCR assays demonstrated more asymmetrical amplification/inhibition with EDTA as inhibitor, with unequal inhibition in reference and transgene reactions, while inhibition was more symmetrical on the ddPCR platform. Finally, a panel of positive reference material with varying GM content from 0.1 to 10% were evaluated on the ddPCR platform and pertinent performance parameters assessed, namely, precision, accuracy and trueness of results, with good performance of the assay.     

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.     

Detection of anisakids in fish and seafood products by real-time PCR

Anisakids are a group of widely distributed nematodes which have acquired high social relevance due to their involvement in foodborne infections caused by consumption of raw or undercooked seafood. A TaqMan^®-LNA probe real-time assay targeting the cytochrome oxidase subunit I (COI) was developed allowing the simultaneous detection of the most important anisakids species present in fish and seafood products.The determination of the detection limit in terms of ppm was 1 ppmFor the validation of method developed, twenty fish and cephalopod samples were experimentally contaminated with anisakid. It was checked that in cases in any anisakids species was present, it was detected because the Ct was always less than 35 and did not produce any case false negatives. The main novelty of this work lies in the fact that it can be applied to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. 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.     

Detection of Roundup Ready soy in highly processed products by triplex nested PCR

Till now, there still has not an effective detection method for the highly processed GM (genetically modified) products. A novel method of the triplex nested PCR, was developed for the sensitive detection of several foreign genes (Lectin, CaMV 35S, CTP, CP4-EPSPS, NOS) in highly processed products. We detected seven representative highly processed products (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, crude soybean oil, soybean refine oil, soybean salad oil) by the triplex nested PCR. The first triplex PCR cannot detect the insert signals in the processed products, and the sensitivity is 0.5%. However the second triplex PCR, which can simultaneously detect RR soybean targets with a sensitivity of 0.005% in the triplex nested PCR. The result indicates the advanced level of the method for the GM products detection. It is a flexible assay to detect the RR soybean in highly processed 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.     

Development of a quantitative real-time PCR assay for viable Salmonella spp. without enrichment

Propidium monoazide (PMA) combined with molecular quantitative real-time PCR (qPCR) has been widely used for only detection of viable bacteria. However, recent studies indicated PMA did not fully inhibit the detection of dead Salmonella. In this study, we developed a more effective PMA Taqman-based qPCR than previous studies to quantify viable Salmonella spp. in raw shrimp. This method has high specificity by using 60 strains belonging to 23 species. The optimization of the PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10⁶ CFU/mL dead cells, while only 10³–10⁴ CFU/mL dead cells could be inhibited in previous reports. This assay could detect viable Salmonella spp. at as low as 36 CFU/mL in pure culture and 100 CFU/g in raw shrimp. By comparing with PMA-qPCR, qPCR and plate counting for quantifying Salmonella in samples, this PMA-qPCR was obviously superior to qPCR and had good agreement with plate counting. In conclusion, this effective method can be used as an available tool to quantify viable Salmonella spp. in raw shrimp.     

Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA

Isogenic NK603 DNA was degraded by sonication or heat and quantified using A₂₆₀ and two fluorescent dye methods. Quantitative PCR (qPCR) experiments were conducted by amplifying an SSIIb-3 endogenous control and an NK603 transgene in untreated, sonicated, and heat treated samples. qPCR reactions on sonicated DNA samples, based on A₂₆₀ quantification, provided 0.125%, 1.14% and 2.15% NK603; while heat treated samples, provided results of 0.128%, 1.42%, and 2.73% NK603. qPCR reactions on sonicated DNA samples, based on the fluorescent dye method, provided results of 0.18%, 0.861% and 1.74% NK603; while heat treated DNA samples, provided results of 0.18%, 1.02%, and 2.16% NK603. The data suggested that fluorescent dye-based quantifications yielded more accurate determinations of the percent genetically engineered (GM) content at higher concentrations, most likely because fluorescent dye quantifications resulted in additional copies of template added into the qPCR. The data in this study suggested that neither fluorescent dye nor spectrophotometric methods of quantification on highly degraded DNA translated into concordant measurements of qPCR amplifiable DNA and accurate C_t values.     

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.     

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.     

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.     

Multi-laboratory evaluation of a PCR method for detection of ruminant DNA in commercial processed animal proteins

In 2013, the European Union (EU) allowed the inclusion of non-ruminant processed animal proteins (PAP) in fish feeds. Plans also exist to allow non-ruminant PAP in poultry and pig feeds but to keep intraspecies recycling forbidden (e.g. poultry to poultry or pig to pig). However, due to the fear of transmissible spongiform encephalopathy (TSE), PAP from ruminant species remains illegal to use in feeds. To ensure feed safety, nations across Europe need to safeguard that commercial fish feeds do not contain PAP from ruminant species. In this work we report a multi-laboratory study on the detection of ruminant PAP in commercial PAP feed ingredients. A total of 19 non-ruminant PAP, provided by the European Fat Processors and Renderers Association (EFPRA), were analyzed for ruminant content with the European Union Reference Laboratory (EURL)-validated PCR assay by three labs and compared to the result obtained with an immunoassay-based method (MELISA-TEK). Ruminant PAP was detected in five of the 19 non-ruminant PAP samples with both methods, but there were methodological and multi-laboratory differences for the PCR method. Compared to the results with the EU-reference laboratory, 3 false-negatives were found by the two other laboratories. We speculate that the discrepancies seen for some of the samples determined with the PCR method rely on the homogenization and grinding step, which is not described in detail in the EURL Standard Operating Procedure (SOP), shift in the normal distribution of Ct-values near the cut-off of the PCR method, or PCR inhibition. In conclusion, this study demonstrates the presence of ruminant DNA in commercially available non-ruminant PAP by use of two methods for the detection of PAP in commercial feed ingredients, and highlights the importance of thorough homogenization prior to DNA extraction and assessment of normal distribution of Ct-values for successful PCR detection at low ruminant DNA contamination levels.     

Detection of Mycobacterium tuberculosis complex in milk using polymerase chain reaction (PCR)

Organisms of the Mycobacterium tuberculosis complex (TBC) are responsible for tuberculosis, a chronic disease which represents a serious public health problem in the world. The most common means of contracting the disease are through inhalation of aerosols, drinking of unpasteurised milk from an infected cow and the sharing of common water or feed sources.In this study a rapid, sensitive and reliable PCR-based method for specific detection of the Mycobacterium TB complex directly from raw milk samples was developed. The DNA extraction method was effective and able to remove inhibitors and allowed the isolation of amplifiable DNA. The procedure demonstrated the presence of the TBC mpb70 gene in 36/50 (72%) of Albanian milk samples tested. The study highlights the need to screen extracommunitarian milk to define an epidemiological background and to guarantee the quality of the raw milk in countries lacking a suitable TB-infection control plan.     

Detection of peanut (Arachis hypogaea) allergens in processed foods by immunoassay: Influence of selected target protein and ELISA format applied

Direct competitive and sandwich ELISA formats developed to determine Ara h1 and Ara h2 proteins were applied in the detection of peanut in model biscuits prepared with a commercial peanut butter as ingredient. The sandwich format for Ara h2 protein could detect the addition of 2.5% peanut butter, whereas the same format for Ara h1 could not detect 5% added peanut. Direct competitive formats for Ara h1 and Ara h2 proteins could detect the addition of 1% and 0.05% peanut butter, respectively. Therefore, competitive format for Ara h2 was selected to be evaluated by four laboratories, obtaining adequate results in term of repeatability and reproducibility. Results obtained indicate that processing decreased the level of extracted proteins and underestimated the amount of Ara h1 and Ara h2 proteins, the effect being more severe for Ara h1. The selection of the target protein and the ELISA format applied greatly influence the detection of peanut in processed foods.