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.     

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.     

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.     

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 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.     

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 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.     

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.     

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.     

Detection of Listeria monocytogenes in raw whole milk for human consumption in Colombia by real-time PCR

The aim of our work was to detect Listeria monocytogenes in raw whole milk from five municipalities of Boyacá-Colombia using real-time PCR. A PCR hybridization probe format was used to analyze the presence of L. monocytogenes, using specific primers to amplify a 149 bp fragment from the metalloprotease gene (mpl). In this study on a total number of 81 samples, 21 gave positive results for L. monocytogenes by real-time PCR and 13 samples were positive by conventional method. These results indicate a high presence of this pathogen in this area of the country and that this method is considerably faster than current standard methods.     

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.     

Simultaneous detection of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in sliced fruits using multiplex PCR

This study aimed to determine the prevalence and quantity of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in sliced fruits from hawker stalls and hypermarkets in Malaysia. Analysis was carried out using the most probable number (MPN) – multiplex polymerase chain reaction (PCR) method. The prevalence of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in 210 samples of sliced fruits examined were 23.3%, 7.6% and 3.8%, respectively with estimated quantity varying from 0 to 19 MPN/g. This study urged the authority to look into the biosafety of sliced fruits in Malaysia.     

Prevalence of Listeria spp. in ready to eat foods (RTE) from Algiers (Algeria)

The aim of this study was to establish the occurrence of Listeria spp., especially Listeria monocytogenes in ready to eat RTE food marketed in Algiers (Algeria).A total of 227 samples were collected from different producers and retailers.All samples were analyzed using a conventional cultivation method AFNOR V08-055.Out of 227 samples tested, 21 (9.3%) tested positive for Listeria spp. among them, 6 (2.6%) tested positive for L. monocytogenes. L. innocua was the most common Listeria species found being detected in 11 samples (4.8%), although both Listeria ivanovii and Listeria welshimeri were detected in 3 (1.3%) and 1 (0.4%) food samples respectively.The study of the antimicrobial sensitivity of Listeria monocytogenes strains showed no resistance.The study has enabled us to detect these contaminants in a wide range of RTE foods, to suggest that contamination likely occurs after heat treatment, and to assess the danger represented by this category of food for populations at risk.     

Ochratoxin A in coffee beans (Coffea arabica L.) processed by dry and wet methods

The incidence of ochratoxin A was studied in different coffee (Coffea arabica L.) samples. A higher incidence of filamentous fungi was observed in the coffee swept from ground and floating coffee samples. The species Aspergillus ochraceus, Aspergillus sulphureus and Aspergillus sclerotiorum were ochratoxin A producing. In 128 (44%) samples ochratoxin A was not detected; however, in 89 samples (31%), ochratoxin A was detected at 0.1–5.0 μg/Kg levels. Other 25% samples presented contamination above 5.0 μg/Kg. This study showed that the fractions coffee swept from ground and floating coffee represents a serious risk of ochratoxin A contamination.     

Tailoring biosensors for Brazil: Detection of insecticides in foods

An acetylcholinesterase (AChE) B multisensor from Nippostrongylus brasiliensis (Nb) was developed, able to detect the most frequently used insecticides in Brazil. The objective was to establish a fast screening-out method, separating the negative samples from the positive ones. The four mutants, which together presented the widest sensitivity spectrum, were: F345A, M301A, W346V and W346A. The combination of these four mutants in a multienzyme biosensor array enabled the detection of 11 out of the 12 most important insecticides at concentrations below 10 μg/kg. The biosensor test was compared with traditional analysis methods, and validated with food samples previously analyzed. The storage stability revealed that the enzyme activity remained stable for 40 weeks; however the sensitivity decreased with time. The biosensor screened out samples with an analysis duration of about 2 h.     

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.     

Prevalence,antimicrobial susceptibility,and molecular characterization by PCR and pulsed field gel electrophoresis (PFGE) of Salmonella spp. isolated from foods of animal origin in San Luis,Argentina

This study was aimed to determine the prevalence of Salmonella spp. in foods of animal origin sold at retail stores over the period 2005–2011 in San Luis, Argentina. Characterization of isolates was performed by biochemical and serological tests, antimicrobial susceptibility assays, detection of invA invasion gene by PCR and comparison of genomic profiles by XbaI DNA restriction and PFGE. Twenty seven Salmonella strains were detected in 27 (6.32%) of 427 samples of foods analysed. Sixteen S. Enteritidis and one S. Montevideo strains from chicken meat (17 positive samples/115 total samples), six S. Anatum strains from pork sausages (6/90), two S. Typhimurium strains from liquid egg (2/60) and two S. Montevideo strains from chicken giblets (2/62) were isolated. No Salmonella strains were recovered from chicken carcasses (0/100). Salmonella strains were susceptible to antimicrobials commonly used for clinical treatment. All isolates carried the invA gene. DNA restriction and PFGE analysis revealed similar genomic profiles within each Salmonella serovar regardless of the food type, sampling year, or retail store where samples were purchased, suggesting the possibility of circulation and transmission of clones of limited diversity in our region.     

An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples

The purpose of this study is to establish a reliable detection method specific for event 98140, a type of multiple herbicide-resistant corn. Based on the 3′- flanking sequence of event 98140, qualitative and quantitative PCR detection assays were developed. The results revealed the LOD of 0.05% of GM maize for qualitative PCR assay and 4 transgenic haploid genome copies for quantitative PCR assay used in this study. The LOQ was 20 transgenic haploid genome copies, and based on this, as low as 0.05% of 98140 genomic DNA could be accurately and quantitatively detected by means of the quantitative method. Two mixed corn samples, with known 98140 contents (2% and 0.5%), were used to verify the developed real-time PCR system, and the expected results were observed. The results indicated that the developed event-specific PCR methods could be used for the identification and quantification of maize line 98140.     

Detection of Escherichia coli O157:H7 in ground beef using immunomagnetic separation and multiplex PCR

In the study 251 fresh ground beef samples sold in Ankara were analyzed to evaluate the prevalence of Escherichia coli O157:H7 by immunomagnetic separation (IMS) based cultivation technique. Virulence factors of the isolates were determined by multiplex PCR. Nineteen (7.6%) of 251 ground beef samples were found as contaminated with E. coli O157. By PCR analyse in two of the isolates fliC_h7 gene was detected and identified as E. coli O157:H7. According to the multiplex PCR one of the isolate has all stx₁, stx₂, eaeA, hly and fliC_h7 genes and the other has stx₁, eaeA, hly and fliC_h7 genes.