Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng 6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng 6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng 6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95 %) between 0.07 and 0.52, a PCR efficiency of 105 % and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng 6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng 6 DNA were observed.     

Effects of processing on detection and quantification of the parvalbumin gene in Atlantic salmon (Salmo salar)

Consumption of Atlantic salmon is a common cause of fish allergies with parvalbumin (Sal s1) being the major allergen. The presence of DNA encoding Sal s1 indicates the presence of Atlantic salmon in food. Using real-time polymerase chain reaction (PCR), the effects of food processing on the ability to detect and quantify the Sal s1 gene were determined. The method was specific for salmon and did not cross-react with 53 other species. Baking and pressure cooking caused a 5–100-fold decrease in detectable copies of the Sal s1 gene. Despite a 98% reduction in detectable copies following pressure cooking for 60 min, the relative standard deviation (RSD) between replicates was 20% and the response was 100-fold grater than the lowest copy number of Sal s1 reliably detected by the assay. Despite efforts to develop a quantitative assay, the PCR assay was qualitative. It is impossible to predict the effects of food matrices not included in this study, some of which may affect the reliability of the assay. Analyses of raw and pressure cooked salmon using a commercial PCR kit indicated comparable results to the PCR assay.     

The effects of blackcurrant powder (Ribes nigrum) supplementation on pasting properties,physicochemical properties,and nutritive values of starch derived from mung bean (Vigna radiata L.) and pea (Pisum sativum L.)

Incorporation of bioactive-containing fruits and/or vegetables into the carbohydrate-rich food matrix are effective strategies to develop food products with functional health benefits. In this study, blackcurrant powder was added into pea starch and mung bean starch to form pastes to investigate the effects of blackcurrant powder on physicochemical and nutritional properties of the starch pastes. The predictive in vitro glycaemic response of the pastes was highlighted by in vitro glycaemic glucose equivalent assay and alpha-amylase inhibitory activity assay. Both assays showed that blackcurrant powder attenuated (P < 0.05) reducing sugar released, through the inhibition of α-amylase. The colour profiles and textural properties of the pastes were modified by the additions of blackcurrant berry powder at different levels. Nutritional characteristics of the pastes, including total phenolic content and antioxidant activity, were significantly changed (P < 0.05) with the addition of blackcurrant powder.     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng?6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng?6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng?6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95?%) between 0.07 and 0.52, a PCR efficiency of 105?% and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng?6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng?6 DNA were observed.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Development and validation of a SYBR‐Green I Real‐Time PCR test to detect bivalves including Mytilus species in foods

The incidence of allergy to seafood, and in particular to molluscs is second only to that of nuts. To protect consumers, the regulators of food products insisted on identifying molluscs as allergens. The aim was to develop quantitative assay for the presence Mytilus species in processed food products. The chosen platform was real‐time PCR (qPCR) targeting either the gene encoding mitochondrial cytochrome C oxidase I or the nuclear gene encoding β‐actin. Recombinant plasmids containing each of target regions were used as a reference for quantification purposes. Limit of detection (LOD) and of quantification (LOQ) were determined. Spiked food samples containing 50–500 μg g^?1 of Mytilus chilensis were analysed both by qPCR and by ELISA. The former assay gave a positive outcome over this range, whereas the latter was sensitive down to a concentration of 125 μg g^?1.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Establishment of a quadruplex real-time PCR for screening of genetically modified tomatoes

With the increase in number of the genetically modified (GM) crops authorized worldwide and specific labeling legislation established by many countries, a reliable and efficient method for routine screening of raw material or processed food products needs to be developed. In this paper, a quadruplex quantitative real-time PCR (qPCR) system is described which allows simultaneous detection of one tomato endogenous gene and three most frequently used transgenic elements in GM products: cauliflower mosaic virus 35S promoter, Agrobacterium tumefaciens nopaline synthase terminator, and neomycin phosphotransferase II gene. The specificities of the assays are optimized and validated. In the quadruplex qPCR system, the detection ranges for all of the four genes were determined to be 8–80,000 copies per reaction. Finally, the established detection system was applied in amplification of exogenous and endogenous genes from 33 raw materials and 35 processed products samples. The results indicate that quadruplex qPCR method is feasible for screening of GM tomato products, even for some processed food. As this detection system could be easily applied to the detection of transgenic elements in other plant species, we expect it will meet the challenges of routine GM crop detection resulting from a rapid increase in the number of GM crops in the future.     

Microsatellite genotyping with HRM (High Resolution Melting) analysis for identification of the PGI common bean variety Plake Megalosperma Prespon

Legumes and common bean (Phaseolus vulgaris L.), in particular, are important crops worldwide, consumed either as dried seeds or fresh fruits. Correct identification of common bean varieties is important, in order to ensure food quality, safety and authenticity for consumers. Recently, DNA based methods, including molecular markers like microsatellites (SSR), have been developed for plant species or variety identification genotyping and for identification of their ingredients in the final food products. Here, we have applied High Resolution Melting (HRM) analysis coupled with four microsatellite markers to facilitate the identification of protected geographic indication (PGI) common bean variety ‘Plake Megalosperma Prespon’ (‘PMP’). The four microsatellite loci used were informative and were used to generate a unique melting curve profile of microsatellites for each variety tested. These microsatellite markers enabled the distinction and identification of the PGI (common bean variety ‘PMP’). Hence, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in common beans.     

Combined effects of matrix and gene marker on the real‐time PCR detection of wheat

DNA‐based methods have been advanced as excellent alternatives for the detection and quantification of gluten‐containing cereals. In this study, it was intended to evaluate the effect of three distinct food matrices on the performance three TaqMan real‐time PCR approaches targeting α2‐gliadin, agglutinin isolectin (Tri a 18) and thioredoxin h(Tri a 25) genes of wheat. The results of wheat flour detection in soya bean, maize and rice model matrices evidenced that sensitivity was considerably affected by both the food matrix and the target gene. Rice matrix allowed the highest sensitivity, in opposition to soya bean. The α2‐gliadin method enabled the best sensitivity, especially when combined with rice matrix (5 mg kg^?1), followed by maize (10 mg kg^?1) and soya bean (50 mg kg^?1). These findings suggest that food matrix effects need to be carefully evaluated when developing real‐time PCR assays for wheat detection/quantification, but without compromising their great effectiveness as tools to monitor gluten‐containing cereals.     

Growth and thermal inactivation of Listeria monocytogenes and Escherichia coli O157:H7 in four kinds of traditionally non-fermented soya bean products

This study evaluated growth and thermal inactivation of Listeria monocytogenes and Escherichia coli O157:H7 inoculated in tofu, dougan, qianzhang and doupi which were stored at 4, 25 and 37 °C and heated at 55, 60, 65 and 70 °C. Growth of the two pathogens in four soya bean products increased with temperature or A_w of soya bean products increasing. At the same temperature, lag time (LT) values of L. monocytogenes (16.32–0.94 h) and E. coli O157:H7 (2.66–0.98 h) in tofu which has the highest A_w were the lowest. When inoculated soya bean products were stored at 4 °C, L. monocytogenes grew slowly, while E. coli O157:H7 did not grow but survived for 14 days. With temperature increasing, δ-values of L. monocytogenes and E. coli O157:H7 in the four soya bean products were decreased. With A_w of soya bean products increasing, thermal resistance of L. monocytogenes decreased, while that of E. coli O157:H7 increased. This study could assist retail soya bean products processors and food industry to enhance safety of soya bean products and design thermal processing regimes.     

DNA extraction and fingerprinting of commercial rice cereal products

DNA was extracted from commercial rice cereal products using modified conventional methods (CTAB, SDS and a commercial kit) in large fragments (>3 kb) and with relatively high yields (1.4–10.7 μg DNA per g of sample) and was used as template for the amplification of a single copy rice gene (i.e. MIPS) fragment (ca. 850 bp) and microsatellite DNAs (ca. 120–400 bp). The cereal products were further discriminated by using six microsatellite markers. The usefulness of DNA analysis was discussed for quality control and authenticity testing of raw rice materials in rice-based food production, and to monitor genetically modified (GM) rice ingredients in commercial food products.     

Specific detection and quantification of Aspergillus flavus and Aspergillus parasiticus in wheat flour by SYBR® Green quantitative PCR

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10⁶ spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.     

Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains

In this study, we investigated the potential of aflatoxin B₁ (AFB1) production by five Aspergillus flavus strains previously isolated from sorghum grains on cereals (barley, maize, rice, wheat and sorghum), oilseeds (peanuts and sesame) and pulses (greengram and horsegram). Five strains of A. flavus were inoculated on all food grains and incubated at 25 °C for 7 days; AFB1 was extracted and estimated by enzyme‐linked immunosorbent assay. All A. flavus strains produced AFB1 on all food grains ranging from 245.4 to 15 645.2 μg kg^?1. Of the five strains tested, strain Af 003 produced the highest amount of AFB1 on all commodities ranging from 2245.2 to 15 645.2 μg kg^?1. Comparatively, the AFB1 accumulation was high on rice grains ranging from 3125.2 to 15 645.2 μg kg^?1, followed by peanuts ranging from 2206.2 to 12 466.5 μg kg^?1. Less AFB1 accumulation was observed in greengram and sesame seeds ranging from 645.8 to 2245.2 and 245.4 to 2890.6 μg kg^?1, respectively. Our results showed that all food grains tested are susceptible to A. flavus growth and subsequent AFB1 production.     

Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the non-ribosomal peptide synthetase (otanpsPN) gene involved in OTA biosynthesis. Seventy five mold strains representing OTA producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1 x 10⁴ to 10 conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between 1 and 10 conidia/g for SYBR Green assay and TaqMan. No significant differences were found between the Ct values obtained from pure mold DNA and pure mold DNA mixed with food DNA. The ability of the designed qPCR methods to quantify two known conidial suspensions inoculated on several foods was evaluated. The amount of conidia assessed by both qPCR methods was close to the inoculated amount for most foods and indicates that the described procedure holds potential for use for the detection and quantification of OTA producing molds in foods.     

Processing parameter optimization for obtaining dry beans with reduced cooking time

The objectives of this study were to investigate the effects of processing beans by soaking and blanching in salt solutions on the quality parameters of the common bean and, in order to obtain a dry product with a reduced cooking time using experimental design techniques. A Plackett and Burman design was used to evaluate the effects of nine factors, soaking, blanching and drying under different conditions, in bean processing. A central composite rotational 2³ design was used with the factors of soaking time, concentration of NaHCO₃ and drying temperature. An increase in the concentration of NaHCO₃ from 0 to 4 g.100⁻¹ mL reduced the cooking time by 10 min. However, increasing the NaHCO₃ concentration increased the level of damage to the product and darkened the bean seed coat, producing greater color differences in the processed bean. The selected conditions for the production of dry quick cooking beans was a time of 13.1 h, a concentration of NaHCO₃ in the soaking solution of 2.3 g.100 mL⁻¹ and a drying temperature of 50 °C. Under the select conditions, it was possible to reduce the cooking time by 53%, with of 5.36 g.100 g⁻¹ of damaged grains and color difference of 8.39.     

Detection of Salmonella spp., Yersinia enterocolitica,Listeria monocytogenes and Campylobacter spp. by real‐time multiplex PCR using amplicon DNA melting analysis and probe‐based assay

Syto9 and probe‐based multiplex real‐time PCR assays for simultaneous detection of a group of foodborne pathogens (named SYLC group), targeting Salmonella spp. (invA gene), Yersinia enterocolitica (ystA gene), Listeria monocytogenes (hly gene) and Campylobacter spp. (rrna gene), have been developed. The Syto9 assay generates amplicon DNA melting curve with four peaks of 86.5 ± 0.5, 84 ± 0.5, 81.5 ± 0.5 and 90.5 ± 0.5 °C corresponding Salmonella spp., Y. enterocolitica, L. monocytogenes and Campylobacter spp. targets, respectively. The sensitivities of the Syto9 and TaqMan assays in artificially inoculated chicken wing rinses were in a range of 3.2 × 10² to 3.1 × 10⁴ and 9.8 × 10² to 1.9 × 10⁴ colony‐forming units per millilitre, respectively, depending on the pathogen. All tested target strains (n = 100) were correctly detected by the both assays, whereas nontarget strains (n = 100) demonstrated no cross‐reactivity representing 100% specificity. The assays are suitable for application in qualitative and quantitative detection of SYLC group pathogens in food matrices.     

Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods

Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography–mass spectrometry (HPLC–MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.