Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach

In 2003 the European Commission introduced a 0.9 % threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5 %. A false-negative rate of only 5 % for 1 % GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.     

Protein quantification, sandwich ELISA, and real-time PCR used to monitor industrial cleaning procedures for contamination with peanut and celery allergens

In the United States, peanut is one of the main sources of food allergens. Similarly, celery is a common allergenic food in Western Europe. Severe allergic reactions to both foods are common. Unexpected allergic reactions can occur after the consumption of celery- and peanut-free foods as a result of inadvertent cross-contaminations during manufacturing. Therefore, in cooperation with a flavor manufacturer, we monitored the cleaning process of slurry preparation equipment with regard to contaminations of follow-up products with celery and peanut compounds. Washing water samples taken after different cleaning steps and follow-up products were analyzed for the presence of celery and peanut traces with a celery-specific real-time polymerase chain reaction (PCR) and a peanut-specific sandwich enzyme-linked immunosorbent assay (ELISA). PCR and ELISA were compared with a nonspecific protein assay to evaluate whether the detection of protein traces can be a fast and cost-effective method for monitoring the effectiveness of wet cleaning procedures. Additionally, the allergenic potential of the celery and peanut mush, which were used as source material, were measured by a mediator release assay using a rat basophilic leukemia (RBL) cell line. In conclusion, the quantification of total protein in washing water was suitable for monitoring the cleaning process. Our study also revealed evidence that, in cases where wet cleaning is applicable, allergenic traces can be removed with high efficiency.     

SYBR green real-time PCR used to detect celery in food

Celery was found to provoke human allergenic response in some countries. Labeling of celery ingredients was required by the European Union, and the threshold set at 10 mg/kg (0.001%). In our study, a celery mannitol transporter (Mat3) gene-based detection method was established by means of SYBR Green real-time PCR technique. No cross-reactivity was found between celery and the other food materials. Absolute detection limit (LODa), relative detection limit (LODr), and practical detection limit (LODp) of the method were determined through experiments on pure celery DNA, DNA mix, and spiked food samples. The method was able to detect 0.001% raw food sample and 0.01% heated food sample. The utility of the method was confirmed by the investigation of 13 commercial foods.     

A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.     

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90–120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.     

Commercialized rapid immunoanalytical tests for determination of allergenic food proteins: an overview

Food allergies have become an important health issue especially in industrialized countries. Undeclared allergenic ingredients or the presence of “hidden” allergens because of contamination during the food production process pose great health risks to sensitised individuals. The EU directive for food labelling lists allergenic foods that have to be declared on food products by the manufacturers. The list includes gluten-containing cereals, crustaceans, eggs, fish, peanuts, soybeans, milk, various nuts (e.g. almond, hazelnut, and walnut, etc.), celery, mustard, sesame seeds, lupin, and molluscs. Reliable methods for detection and quantification of food allergens are needed that can be applied in a fast and easy-to-use manner, are portable, and need only limited technical equipment. This review focuses on the latest developments in food allergen analysis with special emphasis on fast immunoanalytical methods such as rapid enzyme-linked immunosorbent assays (ELISA), lateral-flow immunochromatographic assays (LFA) and dipstick tests. Emerging technologies such as immunochemical microarrays and biosensors are also discussed and their application to food allergen analysis is reviewed. Finally, a comprehensive overview of rapid immunochemical test kits that are currently available commercially is given in tabular form.     

A multiplex, bead-based array for profiling plant-derived components in complex food matrixes

Authentication of processed food ingredients is becoming an important issue for customers, and some DNA-based analytical methods have been developed, especially for animal products. As food products typically contain several different ingredients, a current challenge is to increase the multiplexing capacity of DNA-based methods, to develop “all-in-one” assays. Oligonucleotide-coupled, bead-based suspension arrays are sensitive and reproducible multiplex analytical tools. We applied the Multi-Analyte Profile (xMAP?) technology to develop an assay able to concurrently detect five different plant components in mixed flours and in processed feed and food. Capture probes were targeted to species-specific DNA polymorphisms present within the first intron of plant β-tubulin genes, which can be amplified by the tubulin-based polymorphism-amplification method (TBP-PCR). The workflow is very simple and straightforward, consisting of a PCR amplification step with universal primers, followed by the direct hybridization assay. Results are highly reproducible. For each single plant species, the absolute detection limit was as low as one target DNA copy. In complex mixtures of flours derived from seeds or from commercial dry “pasta,” relative limits of detection ranged, in weight, from 2 % for soybean to less than 0.5 % for wheat. The specificity of the capture probes and the high sensitivity of the method allowed the successful determination of the analytical composition of three feeds as well as eleven food samples, such as snacks, biscuits, and pasta. The multiplexing ability of the assay (up to 100 different analytes) provides scalability and flexibility, in response to specific needs. Schematic representation of the four-step assay for the determination of plant-derived components in food samples

Solution-phase detection of dual microRNA biomarkers in serum

A strategy for the simultaneous detection of multiple microRNA (miRNA) targets was developed utilizing fluorophore/quencher-labeled oligonucleotide probe sets. Two miRNA targets (miR-155 and miR-103), whose misregulation has afforded them status as putative biomarkers in certain types of cancer, were detected using our assay design. In the absence of target, the complementary fluorophore-probe and quencher-probe hybridize, resulting in a fluorescence resonance energy transfer-based quenching of the fluorescence signal. In the presence of unlabeled target, however, the antisense quencher-probe can hybridize with the target, resulting in increased fluorescence intensity as the quencher-probe is sequestered beyond the Förster radius of the fluorescent-probe. The assay design was tested in multiple matrices of buffer, cellular extract, and serum; and detection limits were found to be matrix-dependent, ranging from 0.34 to 8.89 pmol (3.4–59.3 nM) for miR-155 and 2.90–11.8 pmol (19.3–79.0 nM) for miR-103. Single, double, and triple nucleotide selectivity was also tested. Additionally, miR-155 concentrations were assessed in serum samples obtained directly from breast cancer patients without the need for RNA extraction. This assay is quantitative, possesses a low detection limit, can be applied in multiple complex matrices, and can obtain single-nucleotide selectivity. This method can be employed for the multiplex detection of solution-phase DNA or RNA targets and, more specifically, for the direct detection of serum miRNA biomarkers.     

Polymerase chain reaction techniques for food allergen detection

Food allergies represent an important health problem in industrialized countries. Undeclared allergenic foods as contaminants in food products pose a major risk for sensitized persons. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labeling and improve consumer protection. The methods currently used for the detection of potential allergens in foods are to target either the allergen itself or a marker that indicates the presence of the offending food. As markers for the presence of potentially allergenic foods or ingredients, specific proteins or DNA fragments are targeted. In routine food analysis, the enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR) in the form of a real-time PCR or in combination with an ELISA have been used. The availability, the characteristics, and some future aspects of DNA-based methods in the rapid and sensitive detection of potentially allergenic food constituents or contaminations are discussed in this review.     

Allergen immunoassays—considerations for use of naturally incurred standards

The enzyme-linked immunosorbent assay (ELISA) offers many advantages for the detection of potentially hazardous allergenic food residues that might become adventitious components of other foods during the course of food production and processing. ELISAs detect proteins, and food allergens are proteins. ELISAs are sufficiently sensitive and specific for detection of food allergen residues. ELISAs can also be produced in formats that are compatible with the industrial food processing environment. However, ELISAs also have disadvantages that should be carefully evaluated and widely recognized. Various food-processing operations can have profound effects on the detectability of allergenic food residues. ELISAs detect intact proteins but protein hydrolysates evade detection in some ELISA formats. The residual proteins present in some ingredients derived from commonly allergenic sources may also not be easily detected with ELISAs because of the nature of the protein residues remaining, e.g. lipophilic. Processing operations can dramatically lower the solubility of proteins. In some food formulations, heat processing, in particular, induces chemical modifications that can affect antibody binding to epitopes in the ELISA. The use of naturally incurred standards where allergenic food residues are incorporated into various representative food matrices and then processed in a manner similar to “real-world” food processing can reveal some of the limitations of allergen ELISAs. Methods for the preparation of naturally incurred standards in chocolate, cookie, muffin, ice cream, pasta, frankfurter, and cream of potato soup are provided as examples.     

Rapid and Sensitive Detection of <Emphasis Type="Italic">sFAT-1</Emphasis> Transgenic Pigs by Visual Loop-Mediated Isothermal Amplification

Genetically modified (GM) livestock have the potential to contribute to improving the environment and human health, with consumption of fewer resources and reduced waste production. However, the transgene process also poses risks. The safety assessment and control of transgenic animal products have drawn wide attention, and the relevant regulations and technology are being developed. Quick testing technology plays a significant role in on-site and customs sampling. Nowadays, loop-mediated isothermal amplification (LAMP) was widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency and specificity. In this study, a specific, sensitive detection system for detecting sFAT-1 transgenic pigs was designed. A set of six primers including two loop primers was designed for the target sequence. The DNA samples were amplified in less than 1 h at the optimized temperature and detecting by both Nephelometer LA-320c and unaided eyes directly adding calcein. The detection limit of sFAT-1 LAMP was as low as 1.26 ng/μL. Furthermore, blind tests of transgenic and non-transgenic DNA samples were all correctly detected. Hence, the results in this study demonstrated that LAMP is a very useful tool for transgenic detection.     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

Coupled Turbulent Flow Chromatography: LC–MS/MS Method for the Analysis of Pesticide Residues in Grapes,Baby Food and Wheat Flour Matrices

This paper describes an on-line sample preparation method for the simultaneous determination of 48 pesticides in grapes, baby food and wheat flour matrices. Target pesticides were selected to represent a wide variety of chemical structures and three typical matrices were selected. Turbulent flow chromatography was applied for on-line sample cleanup directly coupled to LC–MS/MS. The aim of the method was to reduce total analysis time, eliminate manual laboratory work, provide clean extracts and achieve reproducible results. Single laboratory method validation was conducted establishing limits of detection between 0.8 and 6.0 ng g⁻¹ for baby food, and 0.8–10.3 ng g⁻¹ for other matrices. Within-day precision values varied between 4 and 18 %, while between-day precisions were in the range 5–22 %. Method recovery ranged from 67 to 124 %, and method accuracy was demonstrated by analysis of external quality control samples. The method was also tested on 24 different survey samples from both bio and organic origin. The method was shown to be convenient, fast and fit for purpose in meeting regulatory requirements for pesticide residue monitoring.

     

Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles

This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55 ± 2 nm) through biotin–avidin binding. A second AuNP2 (30 ± 1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents.     

Analysis of Chlorpyrifos in Water,Fruit Juice,and Honeybee Extract by Chemiluminescent Elisa

Abstract

The suitability of competitive enzyme-linked immunosorbent assays (ELISAs) with chemiluminescent detection-based immobilized antigen (indirect assay) for rapid and accurate determination of chlorpyrifos in various food matrices was tested. The limit of detection (LOD) values were 1–1.75 ng mL^?1, the standard curve midpoint (IC₅₀) was 3.5 ng mL^?1, and the assay duration was 1.5 h. Assay application to the analysis of honeybee extract resulted in chlorpyrifos recoveries varying between 62 and 83% in 5–15 ng mL^?1 herbicide concentration range.     

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10⁷ CFU mL^?1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Homogeneous Fluorescence Resonance Energy Transfer Immunoassay for the Determination of Zearalenone

This study demonstrates the use of antigen-antibody binding for the detection of zearalenone. Based on the principle of the fluorescence resonance energy transfer (FRET) phenomenon between antibody and antigen, an immunoassay, in which zearalenone coupled with the anti-zearalenone antibody, was developed, optimized, and applied. Owing to intrinsic fluorescence properties in basic pH conditions with the optimal cationic surfactant, anti-zearalenone and zearalenone played roles as the respective donor and acceptor in the FRET immunoassay. As the concentration of analyte increased, the antigen/antibody emission intensity ratio (I _430 nm/I _350 nm) was enhanced due to larger amounts of zearalenone/anti-zearalenone complexes. This assay, based on the ratio of intensities (I _430 nm/I _350 nm), displayed high specificity and sensitivity with a detection limit of 0.8 ng mL^?1 for zearalenone. The results obtained from analysis of spiked wheat grain samples were found to be in good agreement with those obtained by employing a direct competitive enzyme-linked immunosorbent assay. The label-free, noncompetitive, and homogeneous FRET immunoassay strategy served as a powerful tool for the simple, rapid, and sensitive quantitative determination of zearalenone in food and feed matrices.     

基于高分辨质谱技术的婴幼儿食品中过敏原蛋白质的高灵敏检测

基于高效液相色谱-串联质谱(LC-MS/MS)技术,选择稳定性好、灵敏度高的特征肽段,利用平行反应监测(PRM)技术,实现了多类过敏原蛋白质的高灵敏度同时检测,并成功应用于婴幼儿食品中过敏原成分的分析。对于婴幼儿食品中蛋白质的提取,与传统的丙酮沉淀法比,采用膜上原位样品预处理方法(i-FASP)可实现更高的蛋白质提取效率和抗干扰能力。所检测的过敏原蛋白质的定量限(LOQ)最小可达到0.028 mg/L,其线性范围最宽可跨越4个数量级,且线性关系良好(相关系数R~2≥0.99)。该方法为食品中过敏原蛋白质组学快速分析提供了一种可靠的分析方法。     

Development and validation of a rapid multiplex ELISA for pyrrolizidine alkaloids and their N-oxides in honey and feed

Pyrrolizidine alkaloids (PAs) are a group of plant secondary metabolites with carcinogenic and hepatotoxic properties. When PA-producing plants contaminate crops, toxins can be transferred through the food chain and cause illness in humans and animals, most notably hepatic veno-occlusive disease. Honey has been identified as a direct risk of human exposure. The European Food Safety Authority has recently identified four groups of PAs that are of particular importance for food and feed: senecionine-type, lycopsamine-type, heliotrine-type and monocrotaline-type. Liquid or gas chromatography methods are currently used to detect PAs but there are no rapid screening assays available commercially. Therefore, the aim of this study was to develop a rapid multiplex ELISA test for the representatives of three groups of alkaloids (senecionine, lycopsamine and heliotrine types) that would be used as a risk-management tool for the screening of these toxic compounds in food and feed. The method was validated for honey and feed matrices and was demonstrated to have a detection capability less than 25 μg/kg for jacobine, lycopsamine, heliotrine and senecionine. The zinc reduction step introduced to the extraction procedure allows for the additional detection of the presence of N-oxides of PAs. This first multiplex immunoassay for PA detection with N-oxide reduction can be used for the simultaneous screening of 21 samples for >12 PA analytes. Honey samples (n?=?146) from various origins were analysed for PA determination. Six samples were determined to contain measurable PAs >25 μg/kg by ELISA which correlated to >10 μg/kg by LC-MS/MS.