GHENT UNIVERSITY FACULTY OF PHARMACEUTICAL SCIENCES Department of Bioanalysis Laboratory of Food Analysis Academic year 2015-2016
DETECTION OF SPECIFIC FOOD ALLERGENS IN PROCESSED FOOD PRODUCTS WITH Q-PCR AND ELISA Mohamed BENCHAIB First Master of Pharmaceutical Care
Promoter Prof. dr. De Saeger
Commissioners: Dr. Isabel Taverniers Dr. Marthe De Boevre
GHENT UNIVERSITY FACULTY OF PHARMACEUTICAL SCIENCES Department of Bioanalysis Laboratory of Food Analysis Academic year 2015-2016
DETECTION OF SPECIFIC FOOD ALLERGENS IN PROCESSED FOOD PRODUCTS WITH Q-PCR AND ELISA Mohamed BENCHAIB First Master of Pharmaceutical Care
Promoter Prof. dr. De Saeger
Commissioners: Dr. Isabel Taverniers Dr. Marthe De Boevre
COPYRIGHT "The author and the promoters give the authorisation to consult and to copy parts of this thesis for personal use only. Any other use is limited by the laws of copyright, especially concerning the obligation to refer to the source whenever results from this thesis are cited."
May 31, 2016
Promoter Prof. Dr. Sarah De Saeger
Author Mohamed Benchaib
ABSTRACT Food allergies are a worldwide problem. Up to 5% of the world population has a food allergy. Epidemiological studies reveal that the prevalence of food allergies is still increasing, especially peanut and tree nuts allergies. The symptoms of a food allergy are often mild, with some respiration problems, rash, gastrointestinal symptoms as best known characteristics. Nevertheless, mortality cases are not excluded. Because of the possible life-threatening consequences, the international food authorities require labelled food products. The terms of these labels are well defined in regulations. Fourteen ingredients must be mentioned on the label if present in the food product. Still, 30-40% of the food recalls are due to undeclared food allergens. To detect the allergenic ingredients, sensitive, specific and reliable techniques are required. In food companies ELISA tests and other quick detection methods are often used to identify food allergens. PCR-based methods form a good alternative thanks to their high specificity. Nevertheless, false positive results are not always excluded. Therefore, optimization of the existing methods and development of new methods to detect allergens are required. In 2014 and 2015 some food products containing spices, were recalled from the market place. Peanut and/or almond were detected in these food products. In the first part of this study, we wanted to clarify the question: does the recalled product “chilipepper powder” contain peanut traces, or is the detected signal due to crossreaction from the matrix with the used detection method? For this purpose, bell/chili peppers were tested with a commercial ELISA kit, Ridascreen FAST Peanut, in addition to a peanut-specific real-time PCR, in order to test for possible aspecific reactions or crossreactions from the ELISA/qPCR methods in the matrix (pepper). In the second part of this study a flour matrix, in-house spiked with 9 different nuts, was subjected to different ELISA methods to test (1) the sensitivity of the test methods, by means of a ppm dilution series of the nuts in the flour, and (2) the specificity of the selected methods. In other words: Are the tested ELISA kits as sensitive as they prescribe on the one hand, and do they show possible false positive results for other nuts than they describe on the other hand?
SAMENVATTING Voedselallergieën zijn een wereldwijd probleem. Tot 5% van de wereldbevolking lijdt aan een voedselallergie. Epidemiologische studies tonen aan dat de prevalentie van voedselallergieën nog altijd stijgt, vooral de pinda en boomnoten allergieën. De symptomen van een voedselallergie zijn vaak mild, met ademhalingsproblemen, uitslag en maag- en darmklachten als bekendste kenmerken. Toch zijn sterftegevallen niet uitgesloten. Omwille van de mogelijke levensbedreigende gevolgen, eisen de internationale
instanties
etikettering
van
de
voedselproducten.
De
etiketteringsvoorwaarden zijn te vinden in wetgevingen. Veertien ingrediënten moeten, indien aanwezig in het voedselproduct, vermeld worden op het etiket. Maar nog altijd zijn 30-40% van de teruggeroepen voedselproducten op basis van niet vermelde voedselallergenen. Sensitieve, specifieke en betrouwbare technieken zijn vereist om ingrediënten van allergene afkomst op te sporen. In voedselbedrijven zijn ELISA tests en andere snelle detectiemethodes vaak gebruikt om voedselallergenen op te sporen. PCRgebaseerde methodes zijn goede alternatieve technieken dankzij hun hoge specificeit. Desondanks zijn vals positieve resultaten niet altijd uitgesloten. Daarom zijn optimalisatie van de bestaande methodes en ontwikkeling van nieuwe allergeendetectie methodes vereist. In 2014 en 2015 zijn sommige producten, die kruiden bevatten, teruggeroepen van de markt. Pinda en/of amandelen werden gedetecteerd in deze voedselproducten. In het eerste deel van deze studie, willen we een antwoord bieden op de vraag: bevat het teruggeroepen product “chilipepeper poeder” sporen van pinda, of is crossreactiviteit van de matrix met de gebruikte detectiemethode de oorzaak van het gedetecteerde signaal? Voor deze doelstelling werden paprika’s en chilipepers getest met een commerciële ELISA kit, Ridascreen FAST Peanut, naast een pinda specifieke realtime PCR, om mogelijke aspecifieke reacties of cross-reacties van de ELISA/qPCR methodes in de matrix(peper) te testen. In het tweede gedeelte van de studie werd tarwemeel, intern gespiked met 9 soorten noten, onderworpen aan verschillende ELISA-methodes om (1) de sensitiviteit van de geselecteerde test methodes, via een ppm verdunningsreeks van de noten in de bloem, te testen samen met (2) de specificiteit van de geselecteerde methodes. Met andere woorden: Zijn de geteste ELISA kits enerzijds even gevoelig als beschreven en vertonen ze anderzijds mogelijke valse positieve resultaten voor andere noten.
ACKNOWLEDGEMENTS First of all, I would like to thank my promotor Prof. Dr. S. De Saeger and The Institute of Agriculture and Fisheries Research to give me the opportunity to write this Master thesis. Secondly, I want to thank my experimental mentor Dr. Ir. I. Taverniers helping and guiding me through this project. Answering my endless questions, correcting my thesis and being a true support. Furthermore, I have to mention the laboratory assistants M. Dhondt, A. Staelens and J. Baert. They helped me anytime I needed them with the practical side of my Master thesis. At last, I may not forget the encouragement of my parents. They always push me whenever it’s necessary a little bit further than my limits. My brothers and sister were helping them to making me happier in times it was difficult. I want also to give thanks to my friends who supported and assisted me. They created enough amusement to make sure that I was well rested after work.
Thank you all!
CONTENTS LIST OF ABBREVIATIONS
1
1
INTRODUCTION
3
1.1 FOOD ALLERGY
3
1.2 FOOD ALLERGY AND FOOD INTOLERANCE
4
1.3 MECHANISM
5
1.4 DIAGNOSE ANT TREATMENT
6
1.5 PEANUT AND TREE NUTS ALLERGIES
9
1.6 FOOD ALLERGEN REGULATIONS
10
1.7 FOOD PROCESSING
11
1.8 ALLERGEN DETECTION METHODS 1.8.1 ELISA 1.8.2 PCR 1.8.3 Proteomics 1.8.4 Other detection tests
12 13 14 18 19
2
OBJECTIVES
21
3
METHODS AND MATERIALS
23
4
5
3.1 BELL PEPPERS AND CHILI PEPPERS 3.1.1 Samples and Sample preparation 3.1.2 DNA extraction 3.1.3 DNA concentration measurement 3.1.4 Real-time PCR for peanut 3.1.5 ELISA for peanut
23 23 24 25 26 27
3.2 FLOUR SPIKED WITH 9 DIFFERENT NUTS 3.2.1 Samples and sample preparation 3.2.2 PCR for Pecan and Brazil nut 3.2.3 ELISA for 7 nuts 3.2.4 Modification of the experimental set up of ELISAs for peanut and hazelnut
28 28 29 30 31
RESULTS
32
4.1 BELL PEPPERS AND CHILI PEPPERS 4.1.1. DNA from bell peppers and chili peppers 4.1.2 PCR detection of peanut in pepper 4.1.3 ELISA detection of peanut in pepper 4.1.4 ELISA detection of peanut in rice
32 32 35 36 37
4.2 FLOUR SPIKED WITH 9 DIFFERENT NUTS 4.2.1 DNA concentration measurement and qPCR inhibition test 4.2.2 qPCR detection of pecan in flour
37 37 38
DISCUSSION
40
5.1 BELL PEPPERS AND CHILI PEPPERS
40
5.2 FLOUR SPIKED WITH 9 DIFFERENT NUTS
42
6
CONCLUSION
43
7
ANNEX
45
LIST OF ABBREVIATIONS °C: degree Celsius µL: microlitre APC: Antigen Presenting Cells CFIA: Canadian Food Inspection Agency Cq: threshold of quantification cycle Ct or Cp: threshold cycle DBPCFC: Double Blind Placebo Controlled Food Challenge ddPCR: digital droplet PCR dNTPs: desoxyribonucleotides DSDNA: double stranded DNA ELISA: Enzyme-linked Immunosorbent Assay FDA: Food and Drug Administration FSA: Food Standards Agency g: gram GI: gastrointestinal GMO RM: Genetically Modified Organisms Reference Material IPC: internal positive control LC-MS/MS: Liquid Chromatography-Mass Spectrometer/Mass Spectrometer LOD: Limit of Detection LOQ: Limit of Quantification m/z: mass-to-charge ratio MALDI-TOF: Matrix Assisted Laser Desorption/Ionisation-Time of Flight mg/L: milligrams per litre 1
mg: milligrams min.: minutes ml: millilitre ng/µL: nanogram per microlitre PCR: Polymerase Chain Reaction ppm: part per million PST: Puncture/Prick Skin Test qPCR: real-time Polymerase Chain Reaction RAST: Radioallergosorbent Test rpm: rotations per minute sec.: second ssDNA: single stranded DNA TE: Tris-HCL EDTA
2
1
INTRODUCTION
1.1 FOOD ALLERGY Food allergy can be described as a hypersensitive adverse reaction of the body to a food substance, where IgE antibodies play a major role (1). Food allergens like milk, egg, soy, fish, wheat and even exotic foods like kiwi and papaya can cause a food allergy (2). Most commonly known are peanut and tree nuts allergies. The prevalence of food allergies is estimated between two and five percent of the world population (3, 4). It appears that food allergies are more present in children than adults, with a prevalence about five percent and even higher. This suggests that when children get older they often outgrow their food allergy (4). A hypersensitive person who is allergic to a food substance like peanut, will get an allergic reaction after consuming this substance or food products containing peanut as an ingredient. There are many adverse reactions due to the consumption of ingredients that can cause food allergy. These reactions can differ from mild to severe reactions and include clinical disorders of the gastrointestinal system, the respiratory system and the skin, while anaphylaxis is a rare adverse reaction. This is the most dangerous adverse reaction; the consequences of which can be life-threatening (5).
The reason why some people are allergic to specific foods is completely clear. Some studies suggest that food allergy is genetically transmitted(6). Other studies insinuate that food allergy is a Western wealth disease, where there are more allergic. They support this idea by the fact that Western children from Europe and the United States don’t play outside as much anymore, where their immune system can be triggered by specific substances. This hypothesis can as well be strengthened by the fact that we see more allergic people nowadays than twenty years ago. Adverse parties counter this hypothesis by saying there is more awareness among the people these days and more means that can detect food allergies. It’s also true that children’s immune system and gut barrier is not well developed at young age. In case of the gut flora, new-born mice kept in a germ-free environment are not having a normal tolerance. So they assume that the prevalence of food allergy is not increasing the past few years. It’s the attention towards it and the means researching food allergies that are increased (3).
3
1.2 FOOD ALLERGY AND FOOD INTOLERANCE The words food allergy and food intolerance are commonly confounded with each other. This isn’t strange because both diseases lead to the same symptoms, when eating a food substance there occurs an adverse reaction. The difference is that food allergies are immune-mediated and most of these allergies are IgE-mediated. Food intolerances are not immune-mediated and have or an enzymatic or a pharmacologic or even an undefined cause. In Fig. 1 the taxonomy of adverse reactions used in Europe is illustrated. In the US, no difference is made between toxic and non-toxic adverse reactions. A well-known example of food intolerance is lactose intolerance.
Figure 1: Classification of adverse food reactions in Europe (7)
The difference between milk allergy and lactose intolerance is difficult to distinguish at first sight, as the symptoms are the same. However, if there are IgE-antibodies present in the blood, it’s about milk allergy. But when the enzyme lactase is deficient or even absent, food intolerance can be assigned as disease form (7).
4
1.3 MECHANISM As mentioned above IgE-antibodies are related with food allergies. An allergen has the ability to sensitize the immune system creating IgE-antibodies having a high-affinity for this allergen (8). Sensitization can be described as the interactions between an allergenic (food) protein and cells of the immune system: Antigen Presenting Cells (APCs), T-cells, Bcells. Before the systemic immune system reacts to the allergic food proteins, the physical gut barrier will trap big food proteins to enter the human blood vessel system. Normally, the body develops a tolerance against these food proteins, where intestinal epithelial cells and gut flora play both a major role. Intestinal epithelial cells belong to the APCs, they present an antigen to T-cells and if the T-cells produce a second signal that triggers the T-cells to maturate the result of this process is that B-cells will be excited and produce antibodies which will induce inflammatory responses. When the second signal lacks, there are no antibodies produced and tolerance is developed (3). When allergens are presented for the first time to an allergenic individual, no IgEantibodies will be produced. IgE-antibodies are attained by a second exposure to allergens. This is realised by the class-switching of the B-cells. IgE-antibodies are then produced by the B-cells instead of IgG-antibodies. The IgE-antibodies bind then with mast-cells. Allergens, the antigens, will on their turn bind with the IgE-antibodies. The part of food (glyco)proteins responsible for the interaction with IgE-antibodies are called epitopes. Earlier studies say that epitopes can be either linear or can be conformational. But now, by the information of crystallography, it’s more obvious that (glyco)protein epitopes are all conformational. This can be proved by observing and comparing a free peptide with a peptide as a part of the antigen. A free peptide has a weaker bound with an antibody than a peptide as a component of a complete antigen (9).
The cross-linking of the antibodies linked to their antigen will eventually trigger the mast-cells to degranulate. The result of the cell lysis is the release of inflammatory mediators, like histamine and cytokines. These will affect the GI-tract, the airways and the blood vessels. Increased fluid and mucus secretion, decreased airway diameter, increased blood flow and permeability are characteristics of an allergic reaction. There are acute allergic reactions and late phase reactions. It’s known that histamine plays a major role in the acute phase reaction. 5
Cytokines, chemokines and leukotrienes on the other hand have an important influence on the late phase reactions. The acute response appears within a few minutes and disappears as well in a few minutes. On the contrary a late response occurs after hours and is less wide spread than the acute response. The recovery is also a slow process (11).
Cross-reactions make the mechanism of allergenic reactions even more complicated. When a person is allergic to a substance, reacting the same way to another substance is sometimes possible. This can be declared by homologous proteins which mean that they have identical domains. These domains can bind with the same antibody. Ara h8 is an example of this aspect, this peanut protein is a homologous of the Bet v 1 protein present in birch pollen. Sooner or later, they activate the same antibodies leading to the same symptoms (9, 10).
1.4 DIAGNOSE ANT TREATMENT Diagnosing a person with a food allergy encounters many different methods. First, the examination of the patient and the evaluation of his medical history can already give an idea that a food allergy is present. If an acute reaction like acute urticaria; a skin disorder or in the worst scenario an anaphylaxis occurs, the patient can be questioned what he has eaten in the few past hours. It has to be noted that not only food can trigger an allergic reaction. Animals, dust, pollen… can also provoke an allergic reaction. Secondly, when the thought arises that the symptoms are caused by intake of a food substance, a laboratory evaluation could distinguish between an IgE-mediated reaction and non-immune mediated reaction by looking at the IgE-antibody level in the blood. Therefore, the Radioallergosorbent Test (RAST), is an ideal method. This test detects the blood level of IgE antibodies. As earlier mentioned IgE-antibodies are correlated with an allergic reaction. The Prick/Puncture Skin Test (PST), also an IgE-antibody-based test, uses dilutions of the possible allergen. When these dilutions are applied on the skin and a reaction occurred which is well distinguishable from the negative control, a food allergy can be suspected. But restriction of these tests, the fact that a high IgE-antibody blood level does not always indicate a food allergy, can exist (11).
6
Oral food challenge, also called the DBPCFC, the double blind placebo controlled food challenge, is considered as golden standard for the diagnose of a food allergy. Forasmuch the name claims, individuals are followed by investigators in a strict nutritional therapy, to exclude as much as possible confounding factors; like medications. Food granted allergic for a person is prepared to be unrecognizable for the patient. Therefore, food is being pulverized, freeze-dried and put in the microwave. But then there is still the presence of the typical flavour of the food. By using vehicles, commonly capsules for the food substance and the controls more objective results can be obtained (12, 13).
A new diagnostic tool, ImmunoCap ISAC, the microarray-chip with more than fifty purified food allergens can also be used as a novel diagnostic tool for food allergies. A major advantage is that the amount of serum necessary for this tool is very small in order that this test is an ideal method for diagnosing food allergy in babies/little children. The quality of this test increases when multiple allergens can be distinguished at once. This tool has a comparable or even higher efficiency than the existing diagnostic tools, like PST and ImmunoCap test (Fig. 2), the precursor of the ImmunoCap ISAC test (14).
Figure 2: Principle of the ImmunoCap test (42)
7
Once allergic always allergic? No, there are examples whereat allergic children outgrow their food allergy. Cow’s milk allergy, mostly present in babies not older than one year, is an illustration of this outgrowing phenomenon. Eighty percent of the children will outgrow this intolerance at the age of five. Even cases outgrowing peanut allergy in children are known. Up to twenty percent of the children will develop tolerance against peanut allergy (3).
A food allergy treatment does not exist yet. Individuals suffering from food hypersensitivity are advised to avoid food with ingredients causing the symptoms. Medication used against food allergies are H1 antihistaminics, asthma medications, steroids. These medications work not directly on the cause of the allergy, the proteins of the food substances, but enlighten the appearing symptoms. In case of a prehistory of an anaphylactic shock reaction as a result of food proteins, precaution is an important part of the food management. Prescription of epinephrine and the training of using this medication are also of great importance to avoid any greater complications (15).
8
1.5 PEANUT AND TREE NUTS ALLERGIES Two of the major components in food products that can cause food allergy are peanuts and tree nuts. Around six percent of the world population suffers from a food allergy. About 0.6 percent has a peanut allergy and 0.5 percent has a tree nuts allergy. Both form the main allergen groups causing food allergies in adults. In children, milk and egg (allergens) are the most common products or ingredients causing food allergies followed by peanut. The prevalence of peanut allergy has already doubled in young children the past few years(3, 16).
Table 1. Well-known nuts causing food allergies Peanut Pecan Brazil nut Hazelnut Walnut Macadamia nut Cashew Almond Pistachio
Arachis hypogaea Carya illinoiesis (Wangenh.) K. Koch Bertholletia excelsa Corylus avellana Juglands regia Macadamia ternifolia Anacardium occidentale Amygdalus communis L. Pistacia vera
Peanut generally causes more severe adverse food reactions than any other food substance like tree nuts, eggs, milk. Anaphylaxis is also more common in patients with peanut allergy. A study claims that anaphylaxis is also triggered by other co-factors. Asthma is an example of these co-factors. Of the highest concern is the death as a consequence of a food allergy. Fifty percent and more of the deaths due to food allergies are cases whereby peanut is been taken in (15).
Peanut proteins Ara h1, Ara h2 and Ara h3 are considered to have an important role in peanut allergy. Ara h1 was the first allergen acknowledged as the major peanut allergen. Ara h3 was identified as another major allergen. Ara h2, maybe the most important allergenic protein was granted to have more influence on the human body than the other proteins. This was based on the higher resistance ability of the epitopes of Ara h2 to gastric enzymatic reactions. These findings could suggest that Ara h2 is the most important peanut protein affecting the human body which results in allergic reactions (17). 9
The cross-reactivity in detection methods between peanut and derivative products of spices were recently detected in Europa and North-America. The Food Drug Administration (FDA), the Food Standards Agency (FSA), the Canadian Food Inspection Agency (CFIA) recalled these products after obtaining positive results for the presence of peanut and/or almond. The national authorities also warned the public about these findings and recommended individuals with peanut or tree nuts allergy to be alert by reading the ingredients panel, to avoid ground cumin and seasoning mixes and seek immediate medical care when symptoms occur. Recalling food products which already are on the marketplace happens frequently. (Contaminated) foodstuffs with undeclared food ingredients which have to be mentioned on the label and bacteria in food products belong to the most recalled food products of the market (42-46).
1.6 FOOD ALLERGEN REGULATIONS In the food industry it’s obligatory to mention 14 ingredients if present on the label of their foodstuffs. These 14 ingredients are considered to likely affect a part of the population, causing adverse reactions. Because the consequences of these adverse reactions can be lifethreatening, the European Union had to make some directives regarding this matter. The latest update on regarding certain food ingredients is The Commission Directive 2007/68/EC.
Following ingredients must be indicated on the label: cereals containing gluten, crustaceans, eggs, fish, peanuts, soybeans, milk, nuts, celery, mustard, sesame seeds, sulphur dioxides and sulphites at concentrations more than 10 ppm or 10 mg/L, lupin, molluscs. The category nuts consists of almonds, hazelnuts, pistachio nuts, cashews, macadamia nuts, Queensland nuts, brazil nuts, walnuts and pecan nuts. Not only the pure form of all these food ingredients are mandatory to mention on the label, also derivatives of these products must be indicated. Exceptions on five of these food ingredients are allowed. For example, “nuts used for making distillates or ethyl alcohol of agricultural origin for spirit drinks and other alcoholic beverages” (52).
10
In many foodstuffs none of these described ingredients are normally present. Though, we see that many food labels mention “may contain…”, “made in factory that also handles…” and “made on equipment that also processes…”. The EU does not require these claims, but in the food industries they use these claims as a precaution. Now and again, unnecessary labelling food products with these claims prevents the susceptible customer to purchase a safe product. Nevertheless, hidden allergens can end up in foodstuffs by cross-contamination with allergenic ingredients which are also processed in the same factory. Otherwise, not identifying allergenic ingredients can cause life-threatening situations, even small traces are unsafe for a very susceptible individual. Therefore, appropriate thresholds for allergenic ingredients are difficult to determine, considering the varying sensitivity among the allergenic patients. Hence, reliable, specific and sensitive detection and quantification methods are necessary to accomplish legitimate labelling of foodstuffs (47).
1.7 FOOD PROCESSING Most food products nowadays undergo a number of modifications in factories. There are in general two classes of food processing methods, thermal and non-thermal methods, which can also be combined. Thermal modifications include heating, frying, steaming, boiling, blanching etc. Non-thermal adjustments involve using high pressure, enzymatic modifications, irradiation… An example of a combination of the two methods is autoclaving, combining heat and high pressure at once. These adaptions are mainly induced to improve many aspects of the food, such as the flavour, colour and taste. But they can also be important to the producer, they can make the food more applicable for further modifications (18).
Considering food allergy, a food process method can be an advantage by reducing the allergenicity of food proteins. Sometimes the possibility exists to remove entire proteins of the food products, particularly in case of small proteins. To lose allergenicity denatured proteins must be achieved. This is feasible by adding solvents, extreme pH conditions, intense temperatures and high pressure. Peanut proteins, which can cause a food allergy, can be denatured by frying, roasting, boiling the peanuts. 11
The IgE-level in a peanut allergic person is higher when eating roasted peanuts comparing with the IgE-level after eating fried or boiled peanuts. High IgE-levels are correlated with allergic reactions. Frying and boiling lower the allergenic characteristics of peanut proteins. Especially the Ara h2 and Ara h3 proteins lose most of their allergenicity. Besides, Ara h1 is a more stable protein even at temperatures of 150°C. After the denaturation of Ara h1 a second folding of this protein will result in a structured conformation and will still keep its allergenicity (19). Tannic acid can bind with peanut proteins forming insoluble complexes resulting in failing the bond with IgE-antibodies (20). An enzymatic modification such as a treatment with trypsin leads to a loss in allergenicity of Ara h1 and Ara h3. When adding an autoclaving step after the trypsin treatment a higher loss of the allergenicity was achieved (21).
Tree nut proteins show the same sensitivity to thermal and non-thermal modifications as peanut proteins. It has been described that the allergenic proteins of almond, walnut and cashew are not stable when these nuts are treated non-thermally such as a γ-irradiation treatment. Also thermal treatments show a decrease in the stability of the nuts. Brazil nut and pecan are also responsive to thermal treatments and enzymatic proteolysis (18).
1.8 ALLERGEN DETECTION METHODS Because of the upcoming food allergies and their complications, the food industries are submitted to strict regulations as mentioned above. To detect any potential allergen in the produced food products, they rely on specific methods for detection that are available on the market or are designed within the firm. Test procedures which can be performed in a short time are preferred. Enzyme-Linked Immunosorbent Assays (ELISAs), lateral flow tests and dipsticks are very popular tests to obtain the first information about the presence of an allergic substance. Besides ELISA, Polymerase Chain Reaction (PCR)-based methods can be performed in second instance to have more certainty about the allergen. (Semi)quantification of the allergen can be realised with ELISA- and PCR-based methods.
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1.8.1 ELISA Enzyme-Linked Immunosorbent Assays are accepted as first choice methods to detect allergens. Many reasons contribute to this. First of all, these assays do not need many apparatuses. Secondly, an ELISA is a fast method to obtain results in short time which is necessary in food industries. It has high sensitivity and specificity for detecting allergens. Different types of ELISA exist (23). The simplest format of ELISA is a plate coated with antibodies. The protein solution that consists the antigens is then added. The antigens bind with the antibodies and the remaining binding sites of the antibodies are blocked with another protein, often serum bovine is used in this step. Then other monoclonal antibodies, which also recognize the antigens, are added. These antibodies are linked with an enzyme. After a washing step, a substrate for the enzyme is added and creates a colour density which can be measured by a fluorimeter(22). A more complex, but often used ELISA method is the sandwich ELISA (48). See Fig. 3.
Figure 3: Sandwich ELISA
(1) Wells are coated with an antibody. (2) Sample with antigen is added and binds if recognized by the antibody. (3) Detecting antibody with affinity for the antigen is then added. (4) An enzyme-linked antibody which recognizes the detecting antibody from the previous step is added. (5) A substrate is added and converted by the enzyme from the previous step to a detectable structure (49).
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1.8.2 PCR Polymerase Chain Reaction is a DNA-based method. Where ELISA focuses on the protein itself, PCR will search for DNA sequences which eventually translate into this protein. After extracting the DNA from the food sample, two short oligonucleotide sequences, also called primers, are added to bind to their complementary target sequences. PCR exists of some sequential steps. First, the DNA strands need to divide, this happens at the denaturation temperature of DNA. Often a temperature of 95°C is used. Second, the temperature is decreased to about 55-65°C, the added primers can now anneal to the recognized sequences that translate for the antigen. After elongation with a polymeraseenzyme of the primers with added oligonucleotides, at a standard temperature of 72°C, copies of the target DNA are formed. The denaturation-annealing-elongation process is repeated 30-35 times, to assure enough copies of the target DNA are formed. After the third cycle normally the target DNA is already copied. To visualize these components labelled primers can be used (24).
To analyse the formed PCR products, gel electrophoresis can be used. Gel electrophoresis uses a standard DNA ladder to compare the PCR products with. A well-known used dye is the SYBR Green dye, which makes visualisation possible of the PCR products. The gel in this system is often made of agarose. The DNA fragments achieved in the PCR can be put in the gel in separately. Then an electric field is developed, the negative DNA fragments move from the positive side to the negative side of the agarose gel, which is put in a buffer. Small PCR products will move faster than the larger products. Eventually, the length of the PCR products can be traced. Identification of the formed DNA fragments can be realised by adding a restriction enzyme that recognizes a site on the target DNA (25).
14
Real-time PCR or also called quantitative PCR(qPCR), is another method based on the principles of PCR itself. The main advantage of this method is its higher sensitivity. Only a tiny amount of the PCR products is needed to have results with this method. Real-time PCR follows the amplification of the DNA in time and already in the beginning of the exponential phase, the phase where the first copies are formed, detection is possible. This is illustrated in Fig. 4. Other advantages are the reduced bench time and the risk of contamination that is diminished (26).
Figure 4: Amplification curve of a positive sample (50)
Qualitative and quantitative results can be achieved by using real-time PCR. When the fluorescent signal crosses the background signal i.e. the threshold line, the threshold cycle (Ct or Cp) or the cycle of quantification (Cq) is reached. From this point, an accumulation of the fluorescent signal is associated with an exponential amplification of the DNA with the allergen specific primers. A steady phase is reached when no single stranded DNA fragments can be duplicated.
For quantification of the DNA, standard curves are set up with known DNA concentrations. Each standard has its own Ct value. The higher the DNA concentration, the lower the Ct value. A linear liaison is observed between Ct values and the logarithmic concentration of the DNA. Using this curve, the DNA concentration of the sample of interest, after real-time PCR, can be identified by analysing which logarithmic DNA concentration corresponds with the Ct value of the sample (27, 28). 15
Figure 5: Fluorescence mechanism by using a hydrolysis-based probe (51)
The detection of the amplification products is based on fluorescence. The use of a hydrolysis-based probe is a regularly applied method. The single stranded DNA probe hybridizes with a sequence between the two primers. At one end of the probe a nucleotide is labelled with a fluorescent marker, another nucleotide of the probe is labelled with a quencher. The function of the quencher is to absorb the fluorescence signal emitted by the fluorescent marker. When the DNA polymerase enzyme adds new nucleotides to the primers, at a certain point the probe will normally obstruct the further amplification. But the DNA polymerase enzyme has an extra exonuclease function which disassembles double stranded DNA in its path. The nucleotides of the probe will all be replaced. The nucleotide with the quencher is no longer at a close distance to the fluorescent marker. This makes it possible for the fluorescent marker to emit a signal (29). This mechanism is illustrated in Fig. 5.
It is important to mention that the presence of PCR inhibitors is not a rare phenomenon in real-time PCR. False negative results are possible when these inhibitors are present. These inhibitors can bind to single stranded or double stranded DNA, obstructing the amplification of the antigen sequence. Inhibition of the polymerase enzyme can also appear. Magnesium, a very important co-factor of the polymerase enzyme, can interact with a PCR inhibitor resulting in blocking the formation of the amplicons. 16
Known PCR inhibitors are myoglobin, urea, proteinases, polysaccharides, ethanol, phenol. The use of an IPC, internal positive control, can rule out the presence of a PCR inhibitor. In the same reaction mix, second primers and probe are used to detect a control sequence. When the control sequence is amplified, but the target amplicon is absent, this indicates that the PCR reaction was not blocked by any inhibitors. If the target DNA fragment is not formed and the control sequence is not amplified, PCR inhibitors could have caused these reaction failures (30-34).
More and more applications are published of the digital droplet PCR (ddPCR). This method has a higher sensitivity than real-time PCR. Even the quantification of a DNA sample concentration can be realised without using calibration. The PCR mix, which contains MgCl2, a thermostable polymerase enzyme, a forward and reverse primer, a probe and desoxyribonucleotides (dNTPs) for the amplification of the added DNA, is now partitioned into thousands of droplets by using a water-oil emulsion. The PCR process of denaturationannealing-elongation is now applied on each of these droplets. The following step, the detection is comparable to the detection in the real-time PCR. Measuring the fluorescence of each droplet can predict the presence of the target DNA. At the end an estimation of the concentration of the target DNA can be made. The difference between positive and negative droplets is not always evident. PCR disturbing factors such as PCR inhibitors and delayed PCR onset can also appear. Working on a microscale is very cost efficient, but mistakes are not excluded. Within the process damaged droplets could give false positive and false negative results. False positives will emit a higher fluorescence signal; false negative droplets will emit on their turn a reduced signal. All these factors, which impede the correct analysis of the DNA, are called “rain”. “Rain” is the cause of incorrect determination of concentration thresholds(35). Optimization can be realised by little variations in the temperatures of the PCR process, by variations in the concentrations of the used primers and/or probe(36). Also enzymatic restriction of the DNA, can contribute to more accurate quantification of the DNA(37). Because of the many advantages, ddPCR has been recommended to certify Genetically Modified Organisms Reference Materials (GMO RM) (35).
17
1.8.3 Proteomics Liquid chromatography linked to a tandem of mass spectrometry (LC-MS/MS) is an upcoming method to detect food allergens. LC-MS/MS is less time-saving than PCR of ELISA methods but delivers more reliable results. It does not deal with indirect protein or species DNA identification as ELISA and PCR methods are based upon. In practice the search of conserved peptides present in original and processed food material is the aim of this method (38).
Because of the complexity of food proteins, prefractioning is recommended before MS analysis. Therefore, 2D electrophoresis is proven to be a useful method. Proteins are separated according to their iso-electric point and their molecular weight. These proteins are then subjected to a protease treatment often with the trypsin enzyme. After the digestion step the peptide scan be identified with a mass spectrometer. Usually the protein identification is achieved by MALDI-TOF. A laser collides on the sample creating ionized peptide samples. These peptides travel through a tube and in function of their times of flight a m/z ratio can be set up. Every m/z ratio with its intensity can be assigned to a peptide (39).
A better prefractioning method is liquid chromatography. This strategy can be used to identify the proteins in a straight approach from the complex mixtures when coupled with a mass spectrometer. To accomplish more reliable results and a higher specificity MS tandem mode can be performed. The ionized peptides are passing components which select specific ions for the next MS. These ions are then delayed in speed, fragmented again and then reaccelerated before they enter the next mass spectrometer. A LC-Q/TOF MS/MS already shown its utility by monitoring the peanut proteins Ara h1, Ara h2 and Ara h3. It has to be mentioned that adequate stable peptides are preferred to avoid the effects of processing (40).
18
1.8.4 Other detection tests Lateral flow assays (LFAs) and dipsticks are in addition to the ELISA method also quick screening methods popular in the food industries. In contrast with the earlier described methods, these tests give more qualitative than quantitative results. In food industries they are one of the ideal first test methods to search if any ingredient, that causes an allergy, is present in the produced food product.
Lateral flow devices and dipsticks are actually a simplified form of an ELISA. They are up to three times faster than a classical ELISA test. The best know lateral-flow assay is the pregnancy test. In 1997, the first LFA for peanut was invented. Now, a rapid evolution shows up, more and more LFAs and dipsticks are developed for detecting food components. Fig. 6 represents the different areas on a lateral-flow assay. A lateral-flow assay consists of four compartments. The test sample is applied onto the first compartment. Capillary forces help then move the test sample along the other three compartments. The next compartment incorporates antibodies which can recognize the target allergen.
Figure 6: Compartments of a lateral-flow test (41)
19
These antibodies are usually conjugated with colloidal golden nanoparticles. Further you have the test line, which onto other antibodies are fixed. The antibodies can recognize another part of the target allergen. A positive lateral-flow test gives a coloured test line and a coloured control line. The coloured test line consists of two sort of antibodies, one which is conjugated with the target allergen and the golden nanoparticles and another which binds the previous complex making the test line visible. The control line, the third compartment, rules out false negative results. To obtain a reliable result, the control line has to be coloured. False negative results can be caused by inefficient capillary forces. So, the test sample with the target allergen can’t reach the test line and control line. The control line onto which species specific antibodies are attached is then also not coloured, indicating the test procedure has to be repeated. At the end, an adsorbent pad collects then the rest of the test sample (41)
20
2
OBJECTIVES In 2014 and 2015, in Europe, the United States of America and Canada, certain food
products tested positive for undeclared peanut and/or almond traces. It was remarkable for the FSA, FDA and CFIA that all these food products contained spices. In the UK, a spice mix which was used in three food products was presumed to be the reason. The FDA in America detected undeclared peanuts in products in which ground cumin was processed. In Canada as well, curry powder products tested positive to undeclared peanut and almond.
In the interest of these findings the first part of this study investigates bell peppers and chili peppers with ELISA and qPCR (Fig. 7). The purpose was to study the possible crossreactivity of different detection methods with bell/chili pepper matrices. Both DNA-based methods i.e. real-time PCR, as well as protein-based methods i.e. sandwich ELISA method were used to test this hypothesis. The qPCR and ELISA methods were also tested for their sensitivity and specificity with pure chili pepper reference material spiked with peanut. DNA for the qPCR was extracted with two different kits: The NucleoSpin Food kit and the Qiagen Dneasy Plant Mini kit. The Qiagen method is used as the internal standard kit to extract DNA. By comparing these kits, the kit which can extract a larger amount of DNA and is easier to practice with can be determined. Also for the measurement of the DNA concentration two different methods were applied: spectrophotometric versus fluorimetric measurements. The aim of this part of the work was to choose the most suitable DNA extraction and DNA measurement technique for the particular matrix “pepper”.
In the second part of this study, a wheat flour matrix was spiked with eight different tree nuts and peanut (Fig. 8). A dilution series of all 9 nuts in the flour (from 0 to 100 ppm) was composed. First aim of this part was to evaluate the sensitivity of the AgraQuant (PLUS) ELISA kits by using pure nut dilution series. The influence of wheat flour on the sensitivity of the ELISA kits was a second purpose. The specificity of the ELISA kits was also tested. The last objective was to evaluate the influence of wheat flour and other nuts on the detectability of the target nut.
21
Figure 7: Scheme of objectives for the bell/chili peppers
Figure 8: Scheme of objectives for the nuts-in-flour matrices
22
3
METHODS AND MATERIALS
3.1 BELL PEPPERS AND CHILI PEPPERS 3.1.1 Samples and Sample preparation Nine varieties of the Capsicum annuum-genus (“dolce&piccante”, BLUMEN GROUP SPA, Milan, Italy) that were freshly harvested were collected (Table 3.1). The seeds and mesocarp of the bell peppers and chili peppers were separated and stored at -80°C (Eppendorf Innova Ultra-Low Temperature) (Eppendorf Belgium nv/sa, Rotselaar, Belgium).
Table 3.1 Nine varieties of the Capsicum-annuum genus + pure peanuts VARIETY NAME Bell Pepper PLANET Bell Pepper AURELIO Peperone HABANERO Red Orange Peperone MECHICO Peperone YUCATAN Peperone HABANERO Rosso JALAPENA TURKISH Pepper SPANISH Pepper
ABBREVIATION SEED 1 SEED 2 SEED 3 SEED 4 SEED 5 SEED 6 SEED 7 SEED 8 SEED 9
FLESH 1 FLESH 2 FLESH 3 FLESH 4 FLESH 5 FLESH 6 FLESH 7 FLESH 8 FLESH 9
Peperone CANCUN
SEEDS+FLESH10
Peperone SALTILLO
SEEDS+FLESH11
ROASTED PEANUT
C+1
C+1'
C+1''
SALTED PEANUT
C+2
C+2'
C+2"
For DNA-extraction ground samples were necessary to obtain homogenous samples. The seeds were put in a container and treated with liquid nitrogen (Air Lquide Benelux, Herenthout, Belgium) in order to make them easier to grind. The seed samples were put in the container, liquid nitrogen was added, when the nitrogen is evaporated, the container was put in the ball mill Retsch MM400 (Retsch Technology GmbH, Haan, Germany) and then the time was set on 3 min. and the frequency on 30 times/sec. The mesocarp of the peppers was already been cut in little pieces with the peal on. 23
The flesh of the mesocarp was ground with a machine (SerCoLab, Merksem, Belgium) (Fig. 9) after the samples were put in a plastic sack and put a few seconds in liquid nitrogen. Besides these nine varieties, two extra chili peppers (“dolce&piccante”, BLUMEN GROUP SPA, Milan, Italy) were harvested and dried at room temperature. Seeds and mesocarp were ground as one in a blender with a mini container (Waring commercial blender 8011 ES; MC1 Mini Container) (Waring Commercialer, Torrington, USA) to realize homogenous samples. Afterwards the mixture was ground with the ball mill Retsch MM400 to pulverize the hard seeds, the time was set on 3 min. and the frequency on 30 times/sec. After the homogenous samples were ready, these were stored at -20°C (Liebherr LGex 3410) (Liebherr, Liège, Belgium). Pure peanut samples were acquired from a local store (Colruyt Group, Halle, Belgium). Reference material of pepper (European Spice Services nv, Temse, Belgium) was spiked with pure peanut, acquired from a nut stall, using the Ultra Centrifugal Mill ZM 200 (Retsch Technology GmbH, Haan, Germany).
Figure 9: Grind machine
3.1.2 DNA extraction To assure good quality results in PCR, DNA is first extracted from the samples. This was assured by using two commercial DNA extraction kits, the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and the NucleoSpin Food Kit (Machery-Nagel, Duren, Germany). The procedure starts with the lysis of the cells together with removing the membrane lipids. Then proteins are precipitated by using a protease.
24
The RNA is discharged by adding RNase. If contaminants are still present, these can be removed by the silica columns present in the DNA extraction kits. For the Qiagen extraction 100 mg sample was needed. DNA extraction on the seeds could not be set in double due to the tiny amount obtained from the bell peppers and chili peppers. For the seeds of peperone Habanero Red Orange only 50 mg could be weighed. For the pulp of the peppers, only a few varieties could be weighed in double, for the same reason as for the seeds. Some flesh samples contained a lot of peal, but were included anyway in the test set. According to the protocol of the NucleoSpin Food Kit, 200 mg sample was required. No seeds of the peperone Habanero Red Orange could be analysed with the NucleoSpin Food Kit. A negative control, ultra-pure water (Biochrom GmbH, Berlin, Germany) and a positive control, 400 ppm peanut in pepper, were subjected to these extractions.
3.1.3 DNA concentration measurement To measure the extracted DNA, two methods were used. A spectrophotometric and a fluorimetric
method
were
applied.
To
measure
the
absorbance,
the
UV-VIS
spectrophotometer Biospec Nano (Shimadzu Biotech Europe GmbH, Duisberg, Germany) was used. The fluorescence was measured by the Quantus Fluorometer (Promega Corporation, Madison, USA).
The spectrophotometer was set to detect dsDNA from the DNA solutions obtained from the DNA extraction kits. A tiny amount (2 to 5 µL) of the extracted DNA was pipetted on the reading frame of the Biospec Nano and the path length was set on 0.7 mm.
25
Measuring the DNA extracts was also performed with the Quantus Fluorometer. For each sample 1 µL DNA was added to 199 µL QuantiFluor, which contain a dye that can bind with DNA. The solutions were mixed thoroughly and were incubated 5 min. in the dark. Before measuring the samples, the Quantus Fluorometer was calibrated with a blank and standard sample. The blank and the standard were prepared as followed. To 199 µL QuantiFluor was 1 µL TE buffer added. The standard solution was prepared by adding 1 µL Lambda DNA (400 ng/µL) to 199 µL. These two methods are based on the Beer-Lambert Law:
log I0 / I = ε. l. c with:
I0: intensity of transmitted light I: intensity of incident light ε: molar extinction coefficient (𝐿⁄𝑚𝑜𝑙. 𝑐𝑚) l: path length of sample (cm) c: concentration (𝑚𝑜𝑙 ⁄𝐿)
3.1.4 Real-time PCR for peanut The used primers and probe were based on the study of Bergerova et all.,2011. The peanut specific primers Ara h3 Forward and Ara h3 Reverse (Integrated DNA Technologies, Leuven, Belgium) and peanut specific Ara h3 probe (Eurogentec, Liège, Belgium) were added to the qPCR reaction mix (5x HOT FIREPol qPCR Mix Plus) (Bioconnect, Huissen, Netherlands). The reaction mix also contained DNA, thermostable HOT FIREPol DNA polymerase, dNTPs, MgCl2 and ultra-pure Water. The primer concentrations were 7,5 µM and the used probe concentration was 5 µM. In a volume of 25 µL, the final concentrations of the peanut specific primers were 300 nM and 200 nM for the peanut specific probe. The SEEDS samples were all diluted to 40 ng/µL. For the 400 ppm peanut in pepper samples, tested with the SEEDS samples, a dilution of 40 ng/µL was also evaluated.
26
The conditions for the qPCR for the seeds and mesocarpflesh on the Roche Lightcycler 480II (Roche Diagnostics GmbH, Mannheim, Germany) were based on the data sheet of Solis BioDyne and were set as follows: initial denaturation at 95°C for 15 minutes, 50 cycles consisting of denaturation at 95°C for 15 seconds, hybridization and elongation at 60°C for 1 minute. The cooling procedure was managed at a temperature of 40°C for 10 seconds.
3.1.5 ELISA for peanut A protein-based method is another way to obtain information about the allergenicity of food substances. But bell peppers and chili peppers contain a lot of polyphenols and tannins which could inhibit the detection of peanut. Therefore, the protein preparation starts with adding skim milk powder. Casein, a protein isolated from milk powder, has a high affinity for polyphenols and tannins. That means that as an effect of the addition of skim milk powder, more target proteins will bind with the antibodies attached in the wells. The Ridascreen FAST Peanut (R-Biopharm AG, Darmstadt, Germany) is a sandwich ELISA which can detect peanut specific proteins. Detection of Ara h1 and Ara h2 is achieved with the use of polyclonal antibodies of this ELISA test. The limit of detection (LOD) and the limit of quantification (LOQ) of the Ridascreen FAST Peanut are respectively 1.5 ppm and 2.5 ppm. Different tree nuts like hazelnut, pecan, almond, walnut, cashew and pistachio show no cross-reaction with this test. Also, other food substances such as corn, wheat, sesame or soy do not interfere with the assay. Only cross-reactivity with chickpea (0.2%) and green pea (0.001%) can be observed. The samples underwent a sample preparation as followed. Some samples, only 200 mg was available. For all the SEEDS samples 200 mg was used for the ELISA test, 200 mg of skim milk powder which was manufactured in the Food Pilot division of ILVO (ILVO 370, Melle, Belgium) was added. Then 4 ml diluted extraction buffer (60°C) was added. For Peperone HABANERO Red Orange, no seeds were available for the ELISA test. For JALAPENA, enough sample material was available to evaluate the adapted procedure with the prescribed one.
27
As regards the FLESH samples, for Peperone MECHICO only 200 mg mesocarp was available for the ELISA test. For Peperone Yucatan, no flesh was available for the test. For the samples of which enough sample material, at least one gram, was available, the procedure prescribed in the Ridascreen FAST Peanut kit was used. Next, the samples were intensively mixed with the vortex (VWR International nv/sa, Leuven, Belgium). The alternative procedure was followed by centrifuging 2 ml of the extracts for 10 minutes at 14.000 rpm with the microcentrifuge 5417R (Eppendorf AG, Hamburg, Germany). Hereafter, the ELISA was performed as described in the protocol of Ridascreen FAST Peanut with the use of 100 µL of the supernatant. After adding stop solution, the absorbance was measured with the Fluostar Optima (Isogen Life Science, Temse, Belgium).
3.2 FLOUR SPIKED WITH 9 DIFFERENT NUTS 3.2.1 Samples and sample preparation Wheat flour was obtained from a local store (Colruyt Group, Halle, Belgium). To this flour nine different nuts (Colruy Group, Halle, Belgium) were added and homogenized with the Ultra Centrifugal Mill ZM 200. The eight different tree nuts include pecan, brazil nut, hazelnut, walnut, macadamia nut, cashew, almond and pistachio. Peanut was also added to the wheat flour. Afterwards, a dilution series was made of the flour matrix. The final nuts-inflour concentrations were: 0 ppm, 1.5625 ppm, 3.125 ppm, 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm and 100 ppm. The 25, 50 and 100 ppm samples were diluted after the extraction to 20 ppm with TE buffer (Tris-HCL(1M) and Na2EDTA(0.5M) (Sigma-Aldrich, Machelen, Belgium) because of the quantification limits of the ELISA test kits (Romer Labs Diagnostic GmbH, Tulln, Austria). For pecan and brazil nut only a qPCR method and no ELISA test kit was available to detect these nuts in the spiked wheat flour matrices.
28
3.2.2 PCR for Pecan and Brazil nut The first aim of this part is to examine the flour matrices were examined with different nut-specific ELISA methods to test for the spiked concentrations as well as the tests’ sensitivities. However, not for all 9 nuts there is an ELISA test available (Table 3.2).
Table 3.2: Available commercial ELISA and qPCR kits for peanut and tree nuts Species
peanut hazelnut pecan walnut macadamia para = brazil cashew almond pistachio
qPCR RBiopharm (SureFood)
x* x* x x* x x x x x
qPCR Biotecon (FoodProof)
ELISA RBiopharm (RidaScreen FAST)
ELISA RomerLabs (AgraQuant PLUS)
ELISA Neogen (Veratox)
Lateral Flow test R-Biopharm
x ** x **
x x
x x
x x
x x
x x
x° x
x °°
x
x x x x x x
x x x
**
x
x x x
x
Lateral Flow test Lateral Flow test RomerLabs Neogen (Reveal (AgraStrip) 3-D)
x + pistachio
x x + cashew
x x, x°°° x°°° x°°°
x°°° x, x°°° x°°°
positive control NA Allergen RM800 ** NA NA material available with kit? REMARKS: NA = not available * triplex PCR (+ IAC) also exists ** Allergen RM 800 Allergen RM 800 is a reference material used for the quantitative analysis of allergens in food samples. It contains celery, soy, peanut, hazelnut, walnut and gluten in a defined concentration of 800 mg / kg. The reference material is processed in parallel with the food samples, and the extracted DNA is used to create a standard curve. When used in combination with the foodproof Allergen Detection Kits, quantification of allergenic content in the sample can be ° AgraQuant kit, no PLUS kit °° BioKits Walnut kit, no Veratox kit °°° Reveal for Multi-Treenut
For two nuts, pecan and brazil nut, qPCR was used to detect the nuts in the matrices. For the DNA extractions the SureFood PREP Advanced kit (R-Biopharm AG, Darmstadt, Germany) was used. The extraction protocol of the kit was followed. After the DNA extractions, half of the eluted DNA extracts were purified with the NucleoSpin Gel and PCR Clean-up kit (Machery-Nagel, Duren, Germany), such that higher DNA concentrations can be obtained. A dilution series of the 100 ppm A sample of the spiked flour matrix with concentrations variating from 0.976463 ng/µL to 500 ng/µL was put in the block cycler to test if inhibition can take place.
29
The SureFood Allergen ID Pecan kit and the SureFood Allergen ID Brazil Nut kit (RBiopharm AG, Darmstadt, Germany) were used to make target reaction mixes and inhibition reaction mixes. The dilution series samples were thus pipetted in double, once for the reaction control and once for the inhibition control. The conditions for qPCR were described in the protocol of the SureFood Allergen ID kits. Initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 15 seconds and the ‘hybridization and elongation’ step at 60°C for 30 seconds. The cooling procedure was managed at 40°C for 30 seconds. This process was repeated 50 times. The purified flour matrices after the PCR clean-up, some pure tree nuts samples (pecan A and B, walnut A and B, hazelnut B, pistachio B) and peanut B sample which also underwent the PCR clean-up, were pipetted in the SureFood reactions plate.
3.2.3 ELISA for 7 nuts For seven of the nine nut types an ELISA method with AgraQuant was possible (Table 3.2). For five tree nuts an AgraQuant PLUS sandwich ELISA method can be performed. Walnut detection had to be performed with an AgraQuant Walnut sandwich ELISA, whereby the procedure takes longer than the PLUS kits. One gram from each flour matrix dilution with the 9 different nuts was used. The extractions were examined twice if possible with the ELISA AgraQuant kits. AgraQuant PLUS Peanut and AgraQuant PLUS almond both have a LOD of 0.5 ppm total nut. The other AgraQuant PLUS kits have a LOD of 1 ppm. The quantitation range for all the AgraQuant PLUS kits was 1-25 ppm total nut. The AgraQuant Walnut kit has a LOD of 0.35 ppm and a quantitation range of 2-60 ppm total walnut.
Before the ELISA test, to each sample of one gram, additives 1 and 2 (included in each kit) were added. Then 20 ml hot, not boiling, water was added to each tube. When the tubes were shaken until the additive capsules and sample were dissolved, an aliquot of 2 ml was transferred to a centrifuge tube. After the centrifuging step (12 000 rpm for 5 min.), 150 µL supernatant was used for the ELISA test. The ELISA test was performed as described under “Short Instruction” in the protocol. The absorbance was measured with the Fluostar Optima. 30
3.2.4 Modification of the experimental set up of ELISAs for peanut and hazelnut After carrying out the SureFood qPCR test for pecan, only two different AgraQuant PLUS ELISA kits were evaluated. Peanut and hazelnut were selected to be the allergens of choice. The main aim was still to check the spiked concentrations of the different nuts in the flour, as well as to check the prescribed sensitivities of the kits. However, it was decided to have a more detailed look into this sensitivity on the one hand, and specificity on the other hand. For these purposes four different sub-objectives were formulated. To test the sensitivity of Agraquant PLUS Peanut and AgraQuant PLUS Hazelnut, a dilution series of peanut, as well as for hazelnut in TE buffer was made (from 0.5 to 20 ppm). The sensitivity of the kits was also tested in presence of wheat flour. Only pure peanut in flour dilution series was available (2-4000 ppm), the 20-4000 ppm dilutions were again diluted with TE buffer to remain in the range of the ELISA kit. No dilution series of pure hazelnut in flour was available. The sensitivity of the ELISA kits was also tested for peanut and hazelnut in presence of flour and other nuts. The spiked nut-in-flour dilution series had concentrations from 0 to 100 ppm. Dilutions of 20 ppm were made from the concentrations 25, 50 and 100 ppm with TE buffer. For each previous described undiluted samples one gram was necessary for the ELISA kits. The dilutions with TE buffer were carried out after the protein extraction procedure.
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4
RESULTS
4.1 BELL PEPPERS AND CHILI PEPPERS 4.1.1. DNA from bell peppers and chili peppers DNA was extracted from the peanut in pepper dilution series using the Nucleospin Food kit. It has to be mentioned that the last step, namely the elution of the DNA was performed with a four times smaller elution volume (50 µL) than normally described in the protocol. Afterwards, the DNA concentration was measured with the spectrophotometer Biospec Nano and the Quantus Fluorimeter. Table 4.1.1 shows the obtained DNA concentrations, OD A260/A280 ratios and OD A260/A230 ratios peanut in pepper dilution series samples and the positive control (C1+).
Table 4.1.1 Bell peppers and chili peppers: DNA concentrations, OD A260/A280, OD A260/A230 of peanut in pepper dilution series and pos. control (C+1) after Nucleospin
Nucleospin(200mg) DNA Extraction(PEANUT IN PEPPER)
0 PPM
BiospecNANO(ng/µL)
OD OD Quantus 260/280 260/230 (ng/µL)
A and B
201 202
373,29 371,3 1,98 1,98
0,5 PPM A and B
201 198
360,33 270,69 1,92 1,92
199 198
327,85 402,66 1,92 1,94
198 202
309,17 291,36 1,92 1,93
20 PPM A and B
200 201
262,37 296,71 1,91 1,91
50 PPM A and B
203 200
328,44 316,14 1,94 1,95
100 PPM A and B
203 199
271,70 278,97 1,91 1,96
400 PPM A and B
201 200
237,80 275,22 1,93 1,92
C+1 measured twice
199
702,64 705,74 2,22 2,22
H2O STD-LAMBDA(QUANTUS, 400 ng/µl)
200
1 PPM 5 PPM
32
Weight(mg)
A and B A and B
2,84
2,2
1,55 1,49 1,29 1,27 1,34 1,45 1,29 1,29 1,23 1,33 1,39 1,39 1,19 1,43 1,29 1,32 2,01 2,02 0,71
268 250 280 269 281 319 249 244 210 248 258 253 201 198 197 193 360 0,0096 399
Overall, concentrations were up to 1.5 times higher with the Biospec Nano than with the Quantus Fluorimeter. The pure peanut DNA concentration pointed a major difference between the two methods. The Quantus Fluorimeter measured 360 ng/µL, half of the DNA concentration measured with Biospec Nano. The purity of the DNA extractions could also be measured with the Biospec Nano. The peanut in pepper samples and the pure peanut sample showed OD A260/A280 ratios of at least 1.91, indicating no protein contamination occurred within the DNA extraction procedure. As for other contaminants, the OD A260/A230 ratios of the samples were considered. None of the peanut in pepper samples had an OD A260/A230 ratio higher than 1.8. For the pure peanut sample 2.01 and 2.02 were measured as the OD A260/A230, illustrating no organic compounds or chaotropic contaminants were detected.
For the bell pepper and chili pepper samples two DNA extractions were performed using the Qiagen DNeasy Plant Mini Kit and the NucleoSpin Food Kit. The 400 ppm peanut in pepper dilution samples also underwent these two DNA extractions. In Table 4.1.2A (see Annexed) the DNA concentrations, OD A260/A280ratios and OD A260/A230 ratios after both methods are listed up for the SEEDS samples. Table 4.1.2B shows the negative controls after both methods. Looking at the measurements of the Qiagen extracts, less major differences between the spectrophotometric and fluorimetric method were seen. However, it can be seen that the Quantus Fluorimeter measures almost always lower DNA concentrations, except for the seeds of Bell Pepper PLANET and SPANISH Pepper. The OD A260/A280 ratio was for all samples higher than 1.8, except for the seeds of Peperone HABANERO Red Orange an average optical density of 1.77 was measured. For the OD A260/A230 ratios of the samples, seeds of Bell Pepper AURELIO, TURKISH Pepper and SPANISH Pepper were higher than 1.8. The other samples showed a low optical density ratio, implying contamination of these samples.
After the NucleoSpin extraction, higher DNA concentrations than the Qiagen method were quantified for six samples with the Biospec Nano spectrophotometer. For Peperone HABANERO Red Orange, no seeds were available for the NucleoSpin DNA extraction. For the seeds from Bell Pepper PLANET and SPANISH Pepper, higher DNA concentrations with the Quantus Fluorimeter measuring method were seen.
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The optical density ratios of the wavelengths 260 nm and 280 nm after the NucleoSpin approach outpace the 1.8 ratio limit for all the samples. The OD A260/A230 ratios were only higher than 1.8 for the seeds of Bell Pepper AURELIO and Peperone Yucatan. Generally, differences between spectrophotometric and fluorimetric DNA concentrations are less expressed for the SEEDS samples. It is known that the Biospec Nano spectrophotometer measures all DNA, including ssDNA present in a sample, while Quantus measures only the intact dsDNA. Seeds give more or less pure dsDNA and no ssDNA or degraded DNA nor RNA.
For the FLESH samples, the NucleoSpin Food Kit granted us more DNA than the Qiagen method (Table 4.1.3A). The 400 ppm peanut in pepper samples and negative controls also underwent the two extraction methods (Table 4.1.3B).
All though, very low DNA
concentrations were obtained from the mesocarp samples. The maximum DNA concentration, 19.07 ng/µL, was measured from the SPANISH Pepper after the Qiagen extraction. Bell Pepper AURELIO gave the highest DNA concentration after the NucleoSpin extraction, namely 46.61 ng/µL. There were no noteworthy differences between the spectrophotometric and fluorimetric method after the Qiagen Dneasy Plant Mini Kit. After the NucleoSpin extraction some samples showed a decreased DNA concentration of ¼ when measured with the Quantus Fluorimeter. For other samples Quantus measured about the half of what Biospec Nano measures. The OD A260/A280 ratios varied after the Qiagen DNA extraction around the 1.8 limit. The OD A260/A230 ratios for the mesocarp extracts were very low, so that information about contaminants could not be well defined after the NucleoSpin method.
Also two SEEDS+FLESH samples, Peperone CANCUN and Peperone SALTILLO, were examined. The DNA extraction took place after the homogenization of the seeds and mesocarp as one to evaluate DNA concentrations from seed-mesocarp complex (Table 4.1.4). Again, the NucleoSpin method gives higher DNA yields than Qiagen. Major differences between the two DNA measuring methods are also obvious. The DNA concentrations are up to 2 or 3 times higher with the Biospec Nano spectrophotometer.
34
Overall, the OD A260/A280 ratios of the two samples were higher than 1.8 for but under the ratio of 2.0. Looking at the OD A260/A230 ratio, Qiagen extraction gives too low OD ratios (
