High resolution melting analysis as a new approach to detect almond DNA encoding for Pru du 5 allergen in foods

Almond is responsible for trigging adverse immune responses in allergic individuals, and since it is present in many processed food, it is considered as a potential hidden allergen. Here we propose a novel, simple and highly specific approach to detect almond in a wide range of processed foods. The method consists of a real-time PCR assay targeting the gene encoding for the Pru du 5 allergen in almond, using the fluorescent EvaGreen® dye combined with high resolution melting (HRM) analysis. The new approach allowed the detection of trace amounts of almond down to the level of 0.005% (w/w) and was successfully applied to processed foods. HRM analysis increased the specificity of the assay and was effective in distinguishing almonds from other plant foods, including the closely related fruits from the Rosaceae family. It was demonstrated for the first time that HRM analysis can provide a powerful tool for the identification of allergens in foods.     

Quantitative analysis of shrimp allergen in food matrices using a protein chip based on sandwich immunoassay

Food allergy is increasingly becoming a serious concern these days. With packaged foods becoming the norm of the day, food allergy cases out of accidental consumption are becoming rampant, thereby generating great risks for the subjects involved and prompting food authorities in different countries to formulate new regulations about displaying food allergen data on food labels. Detection of food allergens is conventionally carried out by ELISA or PCR tests. These techniques are limited in that they can only detect one or few allergens at one time. Therefore, in the present study a novel sandwich protein chip assay was developed for quantitation of shrimp allergens in food matrixes. The shrimp allergen model used 3D aldehyde slides as the solid carrier, rabbit antisera as the capture reagent, and biotin-labeled monoclonal antibody as the detector reagent. Resulting antigen–antibody complexes were visualized in the presence of commercial strepavidin labeled with Cy3 to produce fluorescence for quantification. With the LOD of the protein chip being 0.054 mg tropomyosin/kg, the protein chip can quantify down to 0.096 mg tropomyosin/kg. The protein chip was not found to be sensitive to other kinds of foods but cross-reacted to some extent with allergens of some other crustaceans. The recoveries ranged from 69.2 to 99.9%, while the intra- and inter-assay coefficients of variation were <13% and <19%, respectively. It seems that the new assay is reliable enough to detect shrimp allergens in food and food products and help minimize the instances of shrimp allergy. It is also possible to use the protein chip for simultaneous detection of other food allergens.     

食品中杏仁过敏原成分的检测

目的：建立检测食品中桃仁、杏仁过敏原成分的荧光PCR 方法,比较国外3 种ELISA 试剂盒效果。方法：针对杏仁Pru du1 基因设计引物及探针,建立荧光PCR 方法。利用杏仁过敏原参考物质对3 个品牌的ELISA试剂盒的回收率进行比较。结果：建立的荧光PCR 方法,具有很好的特异性;灵敏度为10mg/kg。结论：桃仁及杏仁过敏原成分荧光PCR 检测方法特异性好、灵敏度高,对食品中过敏原的检测有重要的实际意义。     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Short Amplicon-Length PCR Assay Targeting Mitochondrial Cytochrome b Gene for the Detection of Feline Meats in Burger Formulation

Consumption or mixing of feline ingredients in halal and kosher foods is forbidden, and various diseases such as SARS, anthrax, and hepatitis could be transmitted through feline meats. However, since feline species are abundant across the world without any market price and their meats are consumed in exotic foods, the chances of their adulteration in common meats are very high. Several recent reports appreciated short amplicon-length PCR assays for species authentication in processed foods assuming that shorter targets would be thermodynamically more stable than longer ones under natural decomposition and food processing treatments. However, scientific evidence to prove this hypothesis is rarely documented. For the first time, we developed here a PCR assay targeting only a 69-bp site of feline mitochondrial cytochrome b gene, and its authenticity was confirmed by AluI restriction enzyme followed by its separation and detection on a lab-on-a-chip-based automated electrophoretic system. The exceptional target stability was systematically proven over the previously documented shortest target (108 bp) under extreme autoclaving and microwaving treatments both in pure and mixed matrices. The assay specificity was tested against 14 terrestrial and aquatic species commonly consumed in foods, and no cross-species detection was observed. The limit of detection of the assay was 0.1 pg of feline DNA and 0.01 % (w/w) of feline meats in raw meats and cooked burgers, respectively.     

High-throughput detection of spore contamination in food packages and food powders using tiered approach of ATP bioluminescence and real-time PCR

A rapid and quantitative method for detection of Bacillus spores in food/non-alcoholic beverage packages and food powders has been developed using filtration-based ATP bioluminescence and real-time PCR, targeting the sporulation gene (spo0A). In combination with heat activation, the presence and amount of viable bacterial spores (i.e., Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis) was determined within 20 min through ATP signal amplifications. The detection limits of heat activation-ATP bioluminescence assay for B. amyloliquefaciens and B. licheniformis spores on food packages were 1.4 × 10² and 1.0 × 10³ CFU/cm², respectively. In contaminated food powders, B. thuringiensis spores could be detected by the ATP assay within the range of 7.9 × 10⁰ to 3.2 × 10⁴ CFU/mg powder while the PCR detection limit was 614 CFU/mg. Linear relationships between luminescent signal (RLU/mg) and plate count (CFU/mg) were found. The same sample after heat activation-ATP assay could be directly used for real-time PCR as a streamlined detection to confirm the identity of Bacillus spores in food packages and food powders even though some bacterial DNA loss was observed. This tiered approach, filtration-based one-tube ATP luminescence method as a rapid, viable screening and using real-time PCR as confirmation, could serve as a high-throughput tool for the detection of Bacillus spores in the food and beverage industry.     

Aluminium content of some processed foods,raw materials and food additives in China by inductively coupled plasma–mass spectrometry

The level of aluminium in 178 processed food samples from Shenzhen city in China was evaluated using inductively coupled plasma–mass spectrometry. Some processed foods contained a concentration of up to 1226?mg/kg, which is about 12 times the Chinese food standard. To establish the main source in these foods, Al levels in the raw materials were determined. However, aluminium concentrations in raw materials were low (0.10–451.5?mg/kg). Therefore, aluminium levels in food additives used in these foods was determined and it was found that some food additives contained a high concentration of aluminium (0.005–57.4?g/kg). The results suggested that, in the interest of public health, food additives containing high concentrations of aluminium should be replaced by those containing less. This study has provided new information on aluminium levels in Chinese processed foods, raw materials and a selection of food additives.     

基于多重连接探针扩增技术的食品中六重过敏原成分检测

基于多重连接探针扩增（multiplex ligation-dependent probe amplification,MLPA）技术建立了一种六重过敏原成分检测方法,可实现食品中大豆、芝麻、花生、杏仁、榛子和核桃6种成分的同时检测。选取多拷贝ITS基因为靶标基因,设计并合成6组特异性杂交探针。探针经杂交、连接和聚合酶链式反应得到扩增片段,经毛细管电泳分析扩增片段大小可明确区分6种过敏原成分。该体系经20余种相关植物、动物及微生物DNA验证显示其特异性良好,经模拟参考样品验证其检出限为5 mg/kg。30份不同种类市售食品MLPA检测结果表明,该方法性能满足实际样品的过敏原的多重检测。因此,本研究建立的基于MLPA技术的六重过敏原检测方法特异性强、灵敏度高,能够为食品过敏原评估、标识管理和风险控制提供技术支撑。     

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR^® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.     

Effects of food formulation and thermal processing on flavones in celery and chamomile

Flavones isolated from celery varied in their stability and susceptibility to deglycosylation during thermal processing at pH 3, 5, or 7. Apigenin 7-O-apiosylglucoside was converted to apigenin 7-O-glucoside when heated at pH 3 and 100 °C. Apigenin 7-O-glucoside showed little conversion or degradation at any pH after 5 h at 100 °C. Apigenin, luteolin, and chrysoeriol were most stable at pH 3 but progressively degraded at pH 5 or 7. Chamomile and celery were used to test the effects of glycosidase-rich foods and thermal processing on the stability of flavone glycosides. Apigenin 7-O-glucoside in chamomile extract was readily converted to apigenin aglycone after combination with almond, flax seed, or chickpea flour. Apigenin 7-O-apiosylglucoside in celery leaves was resistant to conversion by β-glucosidase-rich ingredients, but was converted to apigenin 7-O-glucoside at pH 2.7 when processed at 100 °C for 90 min and could then be further deglycosylated when mixed with almond or flax seed. Thus, combinations of acid hydrolysis and glycosidase enzymes in almond and flax seed were most effective for developing a flavone-rich, high aglycone food ingredient from celery.     

Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients sesame,almond, lupine and Brazil nut

The inter-laboratory (=ring-trial) validation of 4 food allergen quantification methods using real-time PCR is described. Three single real-time PCR methods for the specific detection and quantification of sesame, almond and Brazil nut were used. Additionally, a multiplex real-time PCR method combining the detection of sesame, almond, Brazil nut and lupine was tested in parallel. Matrix based calibrants (rice cookies) spiked (=incurred) with defined amounts of sesame, almond, lupine and Brazil nut were applied for quantitative evaluation. Cookies based upon wheat and rice flour as well as sauce hollandaise powder each incurred with these allergenic ingredients in the range of 10–123 milligram per kilogram were used as ring-trial samples. The lowest spike level of 10 mg/kg could reproducibly be detected by 6 of 7 PCR systems. In quantitative evaluation of the results, reproducibility standard deviations of approximately 50 % and below were obtained. In addition, the effect of the food matrix on allergen quantification was examined. The range of “recoveries” over all matrices and methods was from 43 to 109 %.     

Application of magnetic polymethacrylate‐based microspheres for the isolation of DNA from raw vegetables and processed foods of plant origin

A method for the microextraction of DNA from raw vegetables and highly processed foods of plant origin suitable for PCR analysis was developed. It is based on non‐selective binding of DNA in the presence of PEG 6,000/NaCl to hydrophilic magnetic nonporous poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) ‐ P(HEMA‐co‐GMA) microspheres covered by carboxyl groups. The quantitative polymerase chain reaction (qPCR) by in‐house designed primers targeting a highly repetitive rDNA locus allowed for detection of plant DNAs in a wide range of concentrations (0.1 pg/μL to 10 ng/μL). The described procedure is fast and simple. We further demonstrate that relatively mild acidic treatment of vegetable at elevated temperatures resulted in a dramatic reduction of PCR efficiency indicating extensive degradation of DNA during pickling. The described method is suitable for the analysis of highly degraded DNA from pickled food products.

Practical applications

DNA‐based methods, such as the polymerase chain reaction (PCR), represent an important genetic approach for identification of plant species composition of foods. DNA from processed foods varies in both quality and quantity between the protocols used. Plant samples are generally characterised by a complex composition containing various inhibitors of PCR. Current methods have been tailored for specific samples while no universal protocol is available. Development of a simple universal procedure leading to PCR‐ready DNA would be beneficial. Isolation strategies based on solid phase systems have become popular for PCR‐ready DNA isolations from complex matrices. Here we applied magnetic hydrophilic P(HEMA‐co‐GMA) microspheres to extract DNA from raw vegetables and highly processed foods of plant origin. Proposed small scale PCR‐ready DNA extraction protocol was comparable with a silica‐based DNeasy Plant Mini Kit (Qiagen) and classical CTAB extraction methods in quality and amplificability of DNA while it is less costly and time consuming.     

Canine-Specific PCR Assay Targeting Cytochrome b Gene for the Detection of Dog Meat Adulteration in Commercial Frankfurters

This report described a cytochrome b (cytb)-based polymerase chain reaction (PCR) assay for the detection of canine tissues in commercial frankfurters. Discriminating detection of canine derivatives in processed food products has important application in halal authentication as well as in health, religions, and fare trades. The assay based on a pair of canine-specific primers that targeted a 100 bp region of canine mithochondrial-cytb gene which is present in multiple copies and highly conserved within the same species. The specificity of the assay was tested against dog and eight most common animal meat species as well as five plant species commonly found in frankfurter formulation. The stability and specificity of the assay were verified under different thermal processing conditions under pure and complex matrices. Three commercial brands of chicken and beef frankfurters were tested in triplicate, and specific PCR products were obtained only from deliberately contaminated formulations. The detection limit of the assay was 0.1 % (0.02 ng DNA) of canine meat spiked with other meats in a typical frankfurter formulation. Shorter amplicon length, superior stability, and higher sensitivity of the assay suggested its potential application in the screening of canine-origin biomaterials in processed food products.     

Simultaneous Analysis of Nε‐(Carboxymethyl)Lysine and Nε‐(Carboxyethyl)Lysine in Foods by Ultra‐Performance Liquid Chromatography‐Mass Spectrometry with Derivatization by 9‐Fluorenylmethyl Chloroformate

Isotope dilution ultra‐performance liquid chromatography–electrospray tandem mass spectrometry with derivatization by 9‐fluorenylmethyl chloroformate was successfully applied to quantify N^ε‐(carboxymethyl)lysine (CML) and N^ε‐(carboxyethyl)lysine (CEL) in processed foods. We demonstrate that this analytical method is well validated for the determination of CML and CEL contents in processed foods. Relative standard deviations (RSD) indicate repeatability (RSD < 6% for CML and CEL) and reproducibility (RSD < 6% for CML and < 7% for CEL) in this method. Percent recovery is also good. We obtain recoveries of 102% to 112% for CML and 86% to 114% for CEL. CML levels detected in the samples vary from 2.29 to 480 mg/kg food, whereas CEL is detected in significantly lower concentrations ranging from 0.56 to 107 mg/kg food. These data could help consumers make better food choices by monitoring intake of advanced glycation end‐products, which may pose a risk to human health.     

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

A highly sensitive real-time PCR system for quantification of wheat contamination in gluten-free food for celiac patients

A quantitative real-time PCR system (Q-PCR) for the detection and quantification of wheat DNA in food for celiacs has been developed and optimised. The DNA was extracted with a new procedure, based on the SDS/Guanidine–HCl/Proteinase K method, but faster, with a higher yield and enough purity to avoid an additional purification step. This is the first report that compares the information provided by a Q-PCR technique with the prolamin levels determined with the most frequently used commercially available ELISA, based on the R5 monoclonal antibody. With the exception of some hydrolysed and highly processed food samples (such as beers, syrups, malt extracts, breakfast cereals…), the rest of the food with prolamin levels above the R5 ELISA quantification limit (1.5 mg/kg) have given positive signals with the Q-PCR system. Moreover, some food samples with prolamin levels below 1.5 mg/kg have also given positive signals with the Q-PCR system, indicating a better sensitivity of the DNA technique. Therefore, the developed Q-PCR system can be used as a non-immunological tool in order to confirm, by the “DNA pathway”, the presence of wheat in food not only for celiacs but also for individuals with wheat allergy.     

A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis

The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml⁻¹, respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.     

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10² CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism.     

A polymerase chain reaction directed to detect wheat glutenin: Implications for gluten-free labelling

Gluten enteropathy or celiac disease (CD) is treated by strict adherence to gluten-free diet for life. Trace amounts of wheat in food from farm to table manifests as a major risk to these individuals. A qualitative polymerase chain reaction method was developed by selectively amplifying a 135 bp fragment of the glutenin gene to detect wheat DNA in a plethora of raw and heat processed foods. The limit of detection was 21.5 pg of DNA. The absence of amplification in other cereals such as oat, rye, barley and maize renders this method exclusive for detection of wheat. The detection of wheat DNA in thermally processed foods by this method, despite extensive DNA fragmentation, evinces the suitability and applicability of the method for labeling gluten-free foods. This method complements the immunochemical methods toward addressing food safety in CD patients and wheat allergics.