Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.     

Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial cytb gene from species of dairy interest

Species identification plays an important role in food allergy prevention and food substitution detection that can reduce the commercial value of a product. For these reasons, many molecular methods have been developed to determine species origin; among them, polymerase chain reaction (PCR)-based methods were successfully applied to processed or unprocessed foodstuffs. An updated PCR-RFLP (restriction fragment length polymorphism) method of the cytb gene was developed for the identification of the 4 species of main interest in the dairy industry (Bos, Ovis, Capra, Bubalus). The comparative analysis of the 92 cytb sequences available in the database belonging to the 4 species allowed identification of 2 highly conserved regions, which were used to design 2 oligonucleotides for the PCR amplification of a 275 base-pair (bp) cytb fragment. The in silico analysis allowed identification of a set of species-specific restriction endonucleases (HaeIII, TaqI, and MwoI), which generated easily analyzable species-specific restriction profiles of the 275 bp cytb DNA fragment. The system was developed for both purified DNA and DNA extracted from meat or dairy products and finally tested on mixed samples, indicating its applicability to foodstuffs.     

Preliminary study on detection of irradiated foodstuffs from the Romanian market

In order to fulfil the European task for market survey in food irradiation the first Romanian laboratory for detection of irradiated foodstuffs was established at IRASM Irradiation Centre. In this preliminary study, a wide range of Romanian food samples (spices, vegetables and meat) gamma irradiated at IRASM have been studied using different detection methods: (1) DNA comet assay, (2) thermoluminescence (TL) and (3) electron spin resonance (ESR) for foodstuffs containing bone or cellulose. The results suggest that there is no general available detection method and there is no perfect detection method. In conclusion, in order to carry out a correct identification of radiation treatment of a food sample it is recommended to use at least two standardised detection methods.     

Detection of Ovine or Bovine Milk Components in Commercial Camel Milk Powder Using a PCR-Based Method

Food ingredient adulteration, especially the adulteration of milk and dairy products, is one of the important issues of food safety. The large price difference between camel milk powder, ovine, and bovine milk powder may be an incentive for the incorporation of ovine and bovine derived foods in camel milk products. This study evaluated the use of ordinary PCR and real-time PCR for the detection of camel milk powder adulteration based on the presence of ovine and bovine milk components. DNA was extracted from camel, ovine, and bovine milk powder using a deep-processed product column DNA extraction kit. The quality of the extracted DNA was detected by amplifying the target sequence from the mitochondrial Cytb gene, and the extracted DNA was used for the identification of milk powder based on PCR analysis. In addition, PCR-based methods (both ordinary PCR and real-time PCR) were used to detect laboratory adulteration models of milk powder using primers targeting mitochondrial genes. The results show that the ordinary PCR method had better sensitivity and could qualitatively detect ovine and bovine milk components in the range of 1% to 100% in camel milk powder. The commercial camel milk powder was used to verify the practicability of this method. The real-time PCR normalization system has a good exponential correlation (R² = 0.9822 and 0.9923) between ovine or bovine content and Ct ratio (specific/internal reference gene) and allows for the quantitative determination of ovine or bovine milk contents in adulterated camel milk powder samples. Accuracy was effectively validated using simulated adulterated samples, with recoveries ranging from 80% to 110% with a coefficient of variation of less than 7%, exhibiting sufficient parameters of trueness. The ordinary PCR qualitative detection and real-time PCR quantitative detection method established in this study proved to be a specific, sensitive, and effective technology, which is expected to be used for market detection.     

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.     

Determination of beta-carbolines in foodstuffs by high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method combined with fluorimetric detection is described for the determination of beta-carboline (norharman) and 1-methyl-beta-carboline (harman). The analysis of foodstuffs for the identification of beta-carbolines is facilitated by clean-up samples using Bond Elut PRS cartridges. Recoveries were excellent. Further, a high-performance liquid chromatographic-mass spectrometric method was also developed for their identification. The concentration of beta-carboline among the foodstuffs and alcoholic beverages varied greatly. Also, norharman and harman were observed in uncooked foodstuffs, whereas acetaldehyde was found in most fermented food. The toxicological implication of beta-carbolines in foodstuffs is discussed.     

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.     

Molecular detection of Burkholderia cepacia in toiletry, cosmetic, and pharmaceutical raw materials and finished products

A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products. Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin. DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35 degrees C. The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B. cepacia. The B. cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene. No DNA amplification was observed in samples that were not spiked with B. cepacia. However, all contaminated samples showed the specific 209-bp fragment for B. cepacia. There was a 100% correlation between standard methods and the PCR assay. Standard microbiological methods required 5-6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h. PCR detection of B. cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products.     

Species-specific identification of ruminant components contaminating industrial crude porcine heparin using real-time fluorescent qualitative and quantitative PCR

Ever since the emergence of bovine spongiform encephalopathy, the source of pharmaceutical heparin has been restricted to porcine intestinal mucosa. In this project, two real-time fluorescent PCR methods were developed to assist with quality control analysis. The first is a qualitative method which relies on SYBR Green I chemistry to confirm the porcine origin of industrial crude porcine heparin (ICPH), identify any ruminant contaminants, and generally control purity. The second is based on TaqMan chemistry and is able to quantitatively identify porcine, bovine, caprine, and ovine components and contaminants in ICPH. By targeting mitochondrial DNA, both PCR systems showed a detection limit of 1 pg DNA and amplification efficiencies ranging between 96% and 102%. Moreover, quantitative PCR showed a detection limit of 0.02 ppm in samples comprising porcine, bovine, caprine, and ovine DNA. The results of qualitative PCR over 27 ICPH samples showed that all samples were porcine in origin and that 17 had ruminant contaminants. The results of quantitative PCR further showed that out of all 17 samples with ruminant contaminants, seven samples had bovine, ovine, and caprine contaminants, two samples had bovine and ovine contaminants, and eight samples had only ovine contaminants. In conclusion, the qualitative PCR system was found to be a relatively inexpensive, rapid, and flexible method of identifying the porcine origin of and ruminant contaminants in ICPH, while the quantitative PCR was found suitable to accurately analyze the components and contaminants in detail. Both methods are suitable for routine control assays for the evaluation of ICPH purity and origins of contaminants.     

BIOTECON diagnostics foodproof Listeria monocytogenes Detection Kit, 5' nuclease in combination with the foodproof ShortPrep II Kit

A method was developed for the detection of Listeria monocytogenes in food. The method is based on real-time PCR using hydrolysis probes (5' Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the BIOTECON foodproof ShortPrep II Kit designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is carried out using the foodproof Listeria monocytogenes Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For the internal comparison study, three different foods (soft cheese, coalfish, and smoked ham) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for detection of L. monocytogenes. From each food, 20 samples were inoculated with a low level (1-10 CFU/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.     

Novel approach for the simultaneous detection of DNA from different fish species based on a nuclear target: quantification potential

The development of DNA-based methods for the identification and quantification of fish in food and feed samples is frequently focused on a specific fish species and/or on the detection of mitochondrial DNA of fish origin. However, a quantitative method for the most common fish species used by the food and feed industry is needed for official control purposes, and such a method should rely on the use of a single-copy nuclear DNA target owing to its more stable copy number in different tissues. In this article, we report on the development of a real-time PCR method based on the use of a nuclear gene as a target for the simultaneous detection of fish DNA from different species and on the evaluation of its quantification potential. The method was tested in 22 different fish species, including those most commonly used by the food and feed industry, and in negative control samples, which included 15 animal species and nine feed ingredients. The results show that the method reported here complies with the requirements concerning specificity and with the criteria required for real-time PCR methods with high sensitivity.     

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.     

Simultaneous determination of bisphenol-A-diglycidyl ether, bisphenol-F-diglycidyl ether, and their derivatives in oil-in-water and aqueous-based canned foods by high-performance liquid chromatography with fluorescence detection

A gradient reversed-phase liquid chromatography with fluorescence detection method for simultaneous identification and quantification of bisphenol-A-diglycidyl ether (BADGE), bisphenol-F-diglycidyl ether (BFDGE), and their 10 derivatives in food matrixes was developed and validated for the analysis of oil-in-water- and aqueous-based foodstuffs. The method linearity range 0.016-10 ppm which are hundred-fold below and tenfold above the EU restriction at 1 ppm (mg/kg). The method detection limits range 0.72-4.20 ppb and the method quantitation limits range 2.40-14.85 ppb, respectively. The validation data indicate excellent precision, acceptable recovery, and good robustness, all supporting a good potential to further develop the method as a standard method for the determination of migrations from interior can coatings into foodstuffs.     

Specific PCR detection of tiger, leopard, and lion ingredients from test samples

A PCR method was developed for specific detection of tiger, leopard, and lion DNA from test specimens for inspection and quarantine or for law-enforced animal protection. Three pairs of specific primers were designed based on the mitochondrial cytochrome b gene of tiger, leopard, and lion and used in the PCR testing. To mimic the effect of food processing on the sensitivity of the test, the tiger muscle and bovine bonemeal powder samples were treated at 133 degrees C for 30 min. At this processing condition, the method was sensitive enough to detect as low as 0.05% of tiger-derived ingredients from the mixed bonemeal powders. The data demonstrate that our PCR method is convenient and economic, with high sensitivity and repeatability, and can be used to detect and identify tiger, leopard, and lion ingredients from various test samples.     

Method of determination of appropriate heat treatment of animal meal by immunoassay developed for detection of cooked beef: interlaboratory study.

An interlaboratory trial was conducted for the validation of an enzyme-linked immunosorbent assay (ELISA) method for determination of appropriate heat treatment of animal meal. A commercially available ELISA test kit developed for the identification of beef in cooked food was used in the study. Twelve laboratories from 7 European countries examined 2 different analytical protocols to establish the most appropriate analytical method. Three different samples were used, 2 animal waste materials sterilized at 129 and 134 degrees C (wet conditions), respectively, and a meat and bone meal material processed at dry conditions (maximum temperature, 140 degrees C). Statistical evaluation applying t-statistics showed that the animal meal treated according to European legislation (>133 degrees C) was clearly distinguishable from the 2 other test materials at a 99% confidence level using both analytical protocols. This method can be considered as a complementary test to the immunoassay developed for the detection of pork in cooked food that is already applied in routine analysis for the surveillance of rendering plants.     

One-PCR-tube approach for in situ DNA isolation and detection

Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field.     

Validation of PCR methods for quantitation of genetically modified plants in food.

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.     

Effective PCR detection of animal species in highly processed animal byproducts and compound feeds

In this paper we present a polymerase chain reaction (PCR)-based method for detecting meat and bone meal (MBM) in compound feedingstuffs. By choosing adequate DNA targets from an appropriate localisation in the genome, the real-time PCR method developed here proved to be robust to severe heat treatment of the MBM, showing high sensitivity in the detection of MBM. The method developed here permits the specific detection of processed pig and cattle materials treated at 134 °C in various feed matrices down to a limit of detection of about 0.1%. This technique has also been successfully applied to well-characterised MBM samples heated to as high as 141 °C, as well as to various blind feed samples with very low MBM contents. Finally, the method also passed several official European ring trials.     

Determination of locust bean gum and guar gum by polymerase chain reaction and restriction fragment length polymorphism analysis

A polymerase chain reaction (PCR) was developed to differentiate the thickening agents locust bean gum (LBG) and the cheaper guar gum in finished food products. Universal primers for amplification of the intergenic spacer region between trnL 3' (UAA) exon and trnF (GAA) gene in the chloroplast (cp) genome and subsequent restriction analysis were applied to differentiate guar gum and LBG. The presence of <5% (w/w) guar gum powder added to LBG powder was detectable. Based on data obtained from sequencing this intergenic spacer region, a second PCR method for the specific detection of guar gum DNA was also developed. This assay detected guar gum powder in LBG in amounts as low as 1% (w/w). Both methods successfully detected guar gum and/or LBG in ice cream stabilizers and in foodstuffs, such as dairy products, ice cream, dry seasoning mixes, a finished roasting sauce, and a fruit jelly product, but not in products with highly degraded DNA, such as tomato ketchup and sterilized chocolate cream. Both methods detected guar gum and LBG in ice cream and fresh cheese at levels <0.1%.     

Sensitive detection and quantification of gliadin contamination in gluten-free food with immunomagnetic beads based liposomal fluorescence immunoassay

Gliadin from wheat is a common food allergen that can induce baker’s asthma, wheat-dependent exercise-induced anaphylaxis, atopic dermatitis, and celiac disease. This gliadin assay focuses on rapidly screen and check for gluten contamination in raw materials and in the gluten-free food production process, not only for wheat-sensitive patients but also for the industries producing gluten-free foodstuffs. The developed assay incorporates the use of anti-gliadin antibody-conjugated immunomagnetic beads (IMBs) to capture the gliadin in samples and fluorescent dyes-loaded immunoliposomal nanovesicles (IMLNs) to produce and enhance the detection signal. Hence, a sandwich complex is formed as “IMBs–gliadin–IMLNs”. Experimental results indicate that this detection platform exhibits good sensitivity for gliadin with a detection limit as low as 0.6 μg mL⁻¹ of gliadin; as the polyclonal antibody showed slight cross-reactions with barley and rye. Excellent recovery rates were found ranging from 83.5 to 102.6% as testing the spiked samples. Moreover, the CV (%) of intra- and inter-assay of this developed assay are 4.8–10.6% and 3.5–9.9%, respectively. Based on a parallel analysis of twenty food samples, the results of this developed assay provide a good consistency with those of an AOAC-approved ELISA kit without any false-negative results. The proposed assay method is thus a highly promising alternative method for detecting the contamination of gliadin in the food industry.