Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

Pork identification in four types of food products, which are sausages and the casings, bread and biscuits, using species-specific polymerase chain reaction (PCR) detection of a conserved region in the mitochondrial (mt) 12S ribosomal RNA (rRNA) gene was developed. Genomic DNA of the food products were successfully extracted except for the casing samples, where no genomic DNA was detected. The extracted genomic DNA was then subjected to PCR amplification targeting the specific regions of the 12S rRNA gene. The genomic DNA from the food products were found to be of good quality and produced clear PCR products on the amplification of 12S rRNA gene of 387 base pairs (bp) from pork species. The species-specific PCR identification yielded excellent results for identification of pork derivatives in food products and it is a potentially reliable and suitable technique in routine food analysis for halal certification.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Comparative study of DNA extraction methods for soybean derived food products

The present work describes the comparison of four DNA extraction methods applied to a wide range of soybean derived food products. The methods included the commercial kits NucleoSpin and GeneSpin, the CTAB, and the Wizard methods. The protocols have been compared for their extraction efficiency, evaluated by the determination of yield and purity of DNA extracts, as well as amplifiability. All the methods produced DNA suitable for PCR amplification for the majority of analysed foods, with the exception of soybean sauces and some processed foods. The NucleoSpin and GeneSpin methods showed the best results for DNA yield and purity, when applied to soybean flours and protein isolates. The CTAB and Wizard methods were generally well suited to all kind the food matrices tested. The extraction of processed foods, such as drinks, desserts or vegetarian foods was better achieved with the CTAB or the Wizard methods as both produced high number of amplifiable extracts. The four methods showed similar performances for real-time PCR amplification.     

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.     

Evaluation of DNA extraction procedures for traceability of various tomato products

Globalization of food trade requires the development of integrated approaches, such as traceability of origin, quality and authenticity, to ensure food safety and consumers satisfaction. In this study, different genomic DNA extraction procedures were evaluated for their applicability to internal traceability of different products in the tomato food chain. Quality, quantity and amplificability by SSR markers of extracted DNA tallied the methods performances; times and costs were considered too. The results were processed with “fuzzy-logic” approach. “Wizard” (Promega) scored the best performance in methods final ranking. This work demonstrated the value of genomic methodologies for internal traceability of tomato-derived goods.     

Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR

The wide application of nucleic acid amplification techniques and the increasing industrial interest toward rapid methods has led to the development and application of PCR based methods for the detection of microbial pathogens in food. In the present paper we describe the development of a multiplex PCR method for simultaneous detection of Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix (liquid whole egg).Four different DNA extraction procedures were evaluated for their application on food and, among these, Chelex resin combined with a DNA purification step were found to better perform on the food system considered.A multiplex PCR system was developed, based on the evaluation and combination of published primer sets, and applied to the simultaneous detection of the target pathogens plus an internal amplification control, both in culture media and in a model food system.The overall system proposed, based on an overnight enrichment step followed by DNA isolation and multiplex PCR, was satisfactorily tested for its specificity and sensitivity and allowed the detection of the presence of bacterial DNA and the identification of the target pathogens down to 10 cells/25 g liquid whole egg.     

Rapid and specific detection of Listeria monocytogenes in smoked salmon with BAX®-PCR

Members of the genus Listeria are ubiquitous, and are therefore also common to the food and the environment. Among them, only Listeria monocytogenes has a pathogenic potential, and can cause infectious diseases (listeriosis) in humans. Conventional microbiological testing methods are labour-intensive and time consuming (4–5 days), and often require a number of different culture media for final isolation and confirmatory tests. In order to overcome these limitations, numerous rapid methods have been developed in recent years. DNA-based methods such as the polymerase chain reaction (PCR) have increasingly been used for rapid and sensitive detection of L. monocytogenes. Among the various available PCR assays, we used the BAX^® system (with two different detection procedures: gel detection and automated detection) to screen for L. monocytogenes in samples of vacuum packaged cold smoked salmon. A total of 27 samples were used for this study. The method was compared to the German standard microbiological detection method according to DIN 11290-1 and -2. Detection of Listeria and L. monocytogenes from salmon samples was performed using Palcam enrichment medium, followed by plating on both Palcam agar and ALOA agar. The BAX^® assay gave identical results for 26 food samples compared to the standard method, including 15 positives. Only in one case the BAX^® system gave a false-positive result, probably due to the amplification of DNA from nonviable cells of L. monocytogenes. In naturally contaminated food samples, the BAX^® method gave good results after 24–48 h. Application of this rapid method is simple and time saving.     

Development and application of DNA analytical methods for the detection of GMOs in food

The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.

Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.

If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.

Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.

Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.     

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.     

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.     

Comparison of implementing HACCP systems of exporter Mexican and Chinese meat enterprises

International trade scrutinises food safety practices and procedures, and food businesses have to review their safety procedures more rigorously. HACCP is increasingly relied on to ensure food safety in both meat-producing and meat-processing enterprises. The objective of this study was to compare incentives, costs, difficulties and benefits of Chinese and Mexican meat-exporting enterprises related to food safety management systems implementation. A questionnaire was applied to identify the main factors involved in HACCP implementation. Data were collected among 32 Chinese and 42 Mexican companies, and analysed using the SAS package. The implementation of the HACCP system was filled out in all exporting meat industries. The results indicated that the major incentives were related to improving product quality for both countries, whilst improving control of the process was the first motivation in the Chinese industry and access to new foreign markets was the first motivation in the Mexican one. In addition, both countries' industries reported that staff training was the most important implementing cost, while product testing was the major operating cost. The difficulties found during HACCP implementation and operation activities were associated with availability of personnel for other tasks for China and costs of certification for Mexico. The reported benefits were relevant for the two countries, due to the ability to reduce microbial counts and increased access to foreign and domestic markets. A better understanding of the costs and benefits associated with HACCP systems could be helpful and is necessary in every segment of the food chain in every sector of domestic and international markets in order to assure food quality and safety.     

A comparative study of DNA extraction methods to be used in real-time PCR based quantification of ochratoxin A-producing molds in food products

Quantification of ochratoxin A (OTA)-producing molds in foods by real-time quantitative PCR (qPCR) may be affected by the DNA extraction method used. In the present work, 6 different methods for extraction of DNA from ochratoxigenic molds in foods were tested. Several combinations of mechanical and thermal lysis of conidia with commercialized DNA extraction kits and enzymatic treatments or resins were evaluated. DNA recovery and quality of extracted DNA was measured by testing the extracted DNA with a conventional PCR and an SYBR Green qPCR amplifying the β-tubulin gene and the non-ribosomal peptide synthetase gene, otanpsPN. Inhibition of conventional and qPCR was not observed when the DNA-extraction method includes an initial thermal disruption of conidia before use of commercialized extraction kit or resin, enzymatic treatment and/or lysis buffer. Of the six methods tested, the one combining thermal lysis of conidia followed by a short enzymatic treatment and incubation with Chelex-100 resin and final extraction with the EZNA kit was selected, since the extracted DNA showed good amplification by conventional PCR for β-tubulin gene and the highest DNA recoveries when tested by qPCR. The method was subsequently validated in different food products such as ripened foods, nuts, and grapes inoculated with Penicillium and Aspergillus species. With this Chelex100-enzymatic-EZNA method good DNA recoveries ranging from 69 to 99% were obtained for all food matrices and fungal species tested. This fast method is a promising tool to be used as routine analysis in HACCP systems in the food industry for quantifying OTA-producing molds by qPCR.     

Fraud identification in industrial meat products by multiplex PCR assay

The identification of animal species used in industrial meat products is very important in respect to economic considerations because European Union, which has implemented a set of very strict procedures to correctly label food. In this paper we present conserved region from mitochondrial 12S rRNA and 16S rRNA genes are powerful region for evaluate the presence of fraudulently added meat in compound food by multiplex polymerase chain reaction assay for the identification of most species (ruminant, poultry and porcine). For each food sources (ground meat, sausages and cold cut) 10 samples were collected and DNA extracted successfully. The results demonstrated that none of the samples were contaminated with porcine residuals, but 40% of sausages samples and 30% of cold cut samples were contaminated with poultry residuals. Also the ground meat samples were not contaminated with poultry residuals.     

Rapid detection of eight vegetable oils on optical thin-film biosensor chips

The development of new and increasingly sophisticated techniques for the authentication of food products continues to be a challenge in public food safety. The authenticity of vegetable oils has become very important as the surge of counterfeit oil into the market increases and consumer confidence decreases. The two methods currently used to authenticate vegetable oils are analysis of species-specific chemical composition by techniques such as chromatography and mass spectrometry, and DNA-based methods such as microarray or qPCR detection. In this study, we have developed a thin-film biosensor chip-based analytical technique for food authentication. Aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface. Biotinylated PCR amplicons were then hybridized with the probes. After wash and brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated PCR chain perfectly matched with the probes can be visualized as a color change on the chip surface (gold to blue/purple). This method could detect trace amount of species-specific DNA from food products and has been demonstrated to be effective with eight different vegetative oils. Our results indicated that this assay is rapid, highly sensitive and specific, and can potentially be used as a high throughput detection method.     

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 of triplex PCR for simultaneous detection of maize,wheat and soybean

A reliable and fast detection of important food plants, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and soybean (Glycine max L.) is of particular interest for food authenticity and safety assessment. In this study, the novel multiplex polymerase chain reaction (PCR) method was developed for the rapid qualitative detection of soybean, maize and wheat. To this purpose, new soybean-specific and maize-specific PCR primers were designed. Their specificity was assayed by uniplex PCRs with different plant species, namely maize, soybean, wheat, oats (Avena sativa), and barley (Hordeum vulgare L). Gel electrophoresis of the amplification products demonstrated high specificity of both primer pairs for identification of relevant species. Subsequently, based on the developed DNA markers, the species-specific triplex PCR targeting maize invertase gene, soybean lectin gene and wheat low-molecular-weight glutenin subunit was developed and optimized for simultaneous identification of these three plant species. The developed PCR method enables specific, effective and rapid detection of maize, wheat and soybean and may be used for food analysis.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

Development of a PCR protocol to detect patulin producing moulds in food products

Patulin is a secondary toxic metabolite with important health effects. Several mould species of Penicillium and Aspergillus genera associated with patulin production have been detected in food products. Thus, specific and sensitive methods to detect patulin producing moulds are needed. The aim of this work was to develop a polymerase chain reaction (PCR) method to detect patulin producing moulds in food. 34 patulin producing and 30 non-producing strains belonging to the main species usually reported in food products were used. Patulin production was firstly evaluated by mycellar electrokinetic capillary electrophoresis and high-pressure liquid chromatography-mass spectrometry in all tested strains. Biosynthesis was also used to develop PCR primers derived from the genes involved in patulin. By means of a primer pair based on the isoepoxydon dehydrogenase (idh) gene, a 496-bp amplicon was specifically detected in all the mould strains previously confirmed as patulin producing, regardless of their genus and species. With the developed method it was possible to detect down to 0.5 ng of pure DNA from producing strains and from 1.8 × 10² to 2.7 × 10³ conidia g^?1 in artificially inoculated foods. No relevant PCR inhibition due to food matrices was observed. The PCR protocol developed could be considered as an appropriate tool to detect patulin producing moulds in food 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.