CN103060462A - Gene chip and detection kit for detecting common food allergens - Google Patents
Gene chip and detection kit for detecting common food allergens Download PDFInfo
- Publication number
- CN103060462A CN103060462A CN2013100192543A CN201310019254A CN103060462A CN 103060462 A CN103060462 A CN 103060462A CN 2013100192543 A CN2013100192543 A CN 2013100192543A CN 201310019254 A CN201310019254 A CN 201310019254A CN 103060462 A CN103060462 A CN 103060462A
- Authority
- CN
- China
- Prior art keywords
- gene
- seq
- primer
- food
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a gene chip and a detection kit for detecting common food allergens, which mainly aim at eight common food allergens of celery, chicken, fish, peanut, sesame, soybean, wheat and almond. The gene chip comprises a solid phase carrier and an oligonucleotide probe which is fixed on the solid carrier and comprises DNA (Deoxyribonucleic Acid) fragments selected from MTD (Maximum Tolerated Dose) gene of celery, mitochondrial DNA of chicken, mitochondrial 16S of fish, Arah1 gene of peanut, Sal gene of sesame, Lectin gene of soybean, GAG56D gene of wheat and Prudul gene of almond. The gene chip and the detection kit can be used for detecting the common food allergens and have the characteristics of being convenience in operation, high flux, high accuracy, high repeatability and the like, and can be used for clinical detection on food anaphylaxis.
Description
Technical field
The present invention relates to a kind of gene chip and comprise the detection test kit of this chip, especially relate to allergen gene chip and detection test kit in a kind of common food.
Background technology
Food anaphylaxis a kind of untoward reaction that to be people produce some food is medically belonging to a kind of transformation reactions.Show that according to some external epidemiology surveys 6%～7% the crowd who accounts for total population 4% and 3 years old Infants Below produces irritated to some food.Infant patient about 80% pair of cow's milk, egg, peanut, wheat, soybean in the time of 5～6 years old produce immunological tolerance, and the allergy of about 20% pair of fish and crustaceans aquatic animal can be eliminated, and be irritated throughout one's life often to remaining.The grownup, the sickness rate of fruits and vegetables allergy becomes zooming trend, mainly is because there is the immunological cross-reaction of fruits and vegetables-pollen.China not yet carries out large-scale inquiry, but among a small circle investigation in early time shows: in Beijing, Guangzhou and Shengli Oil Field, the incidence of resident's food anaphylaxis is 3.4%～5.0%.The clinical manifestation of food anaphylaxis reaction comprises the multi-form clinical symptom such as urticaria, dermatitis herpetiformis, oral allergy syndrome, enteropathy syndrome, asthma and allergic rhinitis, can cause anaphylactic shock, threat to life when serious.The patient tends to cause the disappearance of nutrition absorption in long-term diet allergenic foods process, such as 1～9 years old children's food allergy patients, the intake of calcium, iron, zinc, vitamins D, vitamin-E has 67% less than RDA's.Therefore, food anaphylaxis has been regarded as a kind of serious community nutrition hygienic issues, has caused that the whole world pays close attention to widely.After food anaphylaxis refers to that food advances people's human body, body to generation abnormal immune reaction, cause disorder or the tissue injury of body physiological function, and then cause a series of clinical symptom food allergies and have specificity, various immunological disease physiological mechanisms all can relate to.A common feature of food anaphylaxis reaction is in advance contact and the front contact that sensitinogen must be arranged, and makes body be in sensitization, when again contacting this sensitinogen, just brings out the transformation reactions river.Food anaphylaxis is the most common clinically, one of most important supersensitivity illness.
The food anaphylaxis pathogenesis, the pathogenesis of the food allergy of IgE mediation: food allergen advances to induce in people susceptible person's body the speed that produces immunoglobulin E (IgE) mediation to send out anaphylaxis, its process at first is the special IgE antibody of bone-marrow-derived lymphocyte secretion sensitinogen, the IgE type antibody of sensitization and sensitinogen are at mastocyte and basophilic leukocyte surface commissure, make mastocyte discharge histamine sensitization medium, thereby produce anaphylaxis.Allergic protein has the cog region to T-cell and B-cell, produces narrow spectrum IgE type antibody.Therefore, sensitinogen contains two class antigenic determinants, i.e. T cell antigen determinant and B cell antigen determinant.Antigenic determinant is generally the small peptide less than 16 amino-acid residues.The food allergy of non-IgE type mediation: II, III, the allergy of IV type all can relate to, but still lack direct evidence at present.The thrombopenia that milk brings out can relate to the allergy of II type.Food brings out malabsorption syndrome, food brings out enterocolitis and relates to the allergy of III familial combined hyperlipidemia.The required abundance of food of food allergy induced symptom of non-IgE type mediation is large than IgE type mediation type.
Recently there is the metamorphosis of report part food instead recently to have in the report part food allergy patients serum IgG 4 increase and IgE and other IgG not necessarily increase.Someone thinks that IgG 4 is a kind of allogenic cell chemotactic antibody, can make the basophilic granulocyte release medium, but also have result of study not support above-mentioned discussion under antigen stimulation.The effect of IgG 4 in food allergy there is no final conclusion, remains further to be studied.
The kind of food is thousands of, and wherein only some causes allergy easily.Food of the same clan often has similar sensitization, and is especially more obvious with vegetable food, as the patient of peanut allergy is also had in various degree allergy to other leguminous plant.The food habits of various countries, each department is different, and body also just has corresponding difference to the adaptability of food, thereby the food of sensitization is also different, thinks that such as the westerner mutton seldom causes allergy, but higher than the sensitization of pork at Chinese mutton; The westerner is irritated more to chocolate, strawberry, Fructus Fici etc., then seldom sees in China.According to the data in west, cause that easily irritated food is milk, egg, chocolate, wheat, corn, nuts, peanut, orange, lemon, strawberry, onion, pork, some marine products and fish, clams, turkey and chicken etc.Cause easily that in China irritated food has following a few class:
1. milk and milk preparation are the modal food of infant, also are the common food allergens that brings out infant eczema and asthma.Contain the compositions such as first kind milk-protein, second kind milk-protein and casein in the milk, wherein first kind milk-protein is allergen composition the strongest in the milk.Although first kind milk-protein is after pyroprocessing, its allergenicity can obviously weaken, and still can bring out comparatively serious symptom for the patient of height milk allergy.Think that in the past first kind milk-protein has higher species specificity, so once anti-milk children considered to adopt Goat Milk to substitute, but research in recent years confirms that some antigenicity in the animals milk food of many different generas is the similar children for milk allergy, Goat Milk be not reliable substitute food product particularly to the children of milk severe allergy, should not contain Goat Milk etc. in its food prescription yet.
2. egg and egg-products can bring out all age group patient's allergic symptom, and wherein the Protalbinic acid of egg white is to bring out irritated main component, and yolk then seldom brings out allergy.
3. research has confirmed that shrimps crab class, fish and shellfish etc. all can bring out the partially red fish of flesh of fish color such as allergic symptom, particularly mackerel, trout and salmon and very easily bring out allergic symptom.The Crustachia such as shrimp, crab sea-food also contains the allergen Chang Naire of higher these foods of allergen composition, and prepared food also usually brings out allergy.
4. oil crops such as peanut, sesame and cottonseed: mainly to contain higher albumen relevant with these foods, in case make oil prod then seldom bring out allergy.Often can run into clinically the patient of peanut Anaphylactic shock attributed and asthma.Soya bean, mung bean, red bean, black soya bean etc. all can bring out allergy.
5. baker's asthma (Baker.s asthma) is namely relevant with contact flour.Nuts comprises English walnut, hickory, Pistacia vera, fibert, pine nut and chestnut etc., vegetables comprise kidney bean, green soya bean, Lupinus albus, mushroom, tomato, capsicum, leek, garlic, eggplant, Chinese cabbage and fiddlehead etc., and fresh fruit such as peach, apple, banana, strawberry, pineapple, plum, cherry and coconut etc. usually can bring out allergic symptom.Meat and meat product thereof: common beef, mutton, pork, chicken, meat, rabbit meat and the duck etc. of comprising, uncommon meat comprises that dog meats, goose, soft-shelled turtle meat and bird meat etc. also can bring out allergy, have often occurred in the syndromic patient of bird egg such as the allergy to bird meat.The healthcare products that coffee, beer, grape wine, liquor, pollen are made, chocolate and some edible insect (such as locust, silkworm, pupa and bean worm etc.) all can be brought out allergic symptom.
Current foodstuff anaphylactogen detection method mainly contains Immunological Method and nucleic acid amplification, yet no matter ELISA method or nucleic acid detection method, usually can only detect simultaneously a kind of anaphylactogen, and in actual food product detects, need to carry out examination to anaphylactogen and detect, namely multiple anaphylactogen be detected simultaneously.Develop rapidly along with molecular biological, increasing food genomic nucleic acid sequence comes forth, and people are deep into nucleic acid, gene level from external morphological structure and biochemical characteristic to the understanding of food allergen.On this basis, people have set up many new detection techniques, such as nucleic acid probe (Nuclear acid probe) and polymerase chain reaction (Polymerase chain reaction, PCR) etc., because have highly sensitive, high specificity, easy and simple to handle, the characteristics such as reaction is quick, oneself progressively is applied to the detection of anaphylactogen in the food.Under such background, biochip technology arises at the historic moment.The development of molecular Biological Detection technology provides good technology platform for the anaphylactogen rapid detection.At the anaphylactogen detection field, all set up at present the detection technique of many PCR-based methods both at home and abroad, but because round pcr can only be for a certain or several anaphylactogens, and the anaphylactogen kind that exists in the food is a lot, this detects most of common food allergenss simultaneously with regard to needs, this limitation of PCR has increased the workload that detects greatly, is not suitable for the rapid detection of a large amount of samples in field such as food allergens analysis.And biochip technology provides a kind of strong technology platform take its high-throughput, high specific, high responsive, easy and simple to handle, the characteristics of reacting fast as the anaphylactogen detection.For this reason, among the present invention take DNA as detecting target, choose 8 kinds of foodstuff samples, set up a kind of for detection of anaphylactogen novel gene chip in the food, time-consuming, the consumption power that exist to remedy the traditional detection technology, the defective that resolving power is poor, the expansion sensing range, improve detection sensitivity and specificity, reduce labour intensity, shorten sense cycle.
Summary of the invention
Gene chip of the present invention comprises solid phase carrier and is fixed on oligonucleotide probe on this solid phase carrier, and this oligonucleotide probe comprises at least a in the following dna fragmentation:
1. the MTD gene of celery
2. the Mitochondrial DNA of chicken
3. the Mitochondrial DNA of fish
4. the Arah1 gene of peanut
5. the Sal gene of sesame
6. soybean (bean sprouts) Lectin gene
7. the GAG56D gene of wheat
8. the Pru dul gene of almond
Described oligonucleotide probe preferably has the nucleotide sequence shown in the SEQ ID NO:1-8, or is different from SEQ ID NO:1-8 but the coded identical nucleotide sequence of aminoacid sequence of aminoacid sequence and the SEQ ID NO:1-8 of coding.
Above-mentioned preferred sequence and function are as follows:
SEQ ID (5'-3')
NO:1 ATTACATGCTGAGTCACGATGAGCGTG is for detection of the celery anaphylactogen;
NO:2 TTTACGACCTCGATGTTGGATCAGGAC is for detection of the fish anaphylactogen;
NO:3 AGCTCAGGAACTTGCCTCAACAGTGCG is for detection of Peanut Allergen;
NO:4 TTCCCGCAGCCCCAACAACCGCAACAA is former for detection of Wheat Dood Allergy;
NO:5 TCGCAGGTGCAACATGCGACCCCAGCA is for detection of the sesame anaphylactogen;
NO:6 CCTTTCATCCTCATTTCTATGAATCCG is for detection of the chicken anaphylactogen;
NO:7 CACAAAAAGGCTTGCAGATGGGCTTGC is for detection of original soybean sensitive;
NO:8 GAGAAAATCAGTTATGAGATTAAGTT is for detection of the almond anaphylactogen;
Gene chip of the present invention can be used for the detection of common food allergens celery, chicken, fish, peanut, sesame, soybean, wheat, almond.
The preparation method of gene chip of the present invention mainly comprises step:
1) according to the nucleotide sequence shown in the SEQ ID NO:1-8, according to the allergen gene in celery, chicken, fish, peanut, sesame, soybean, wheat, almond design and prepare primer for pcr amplification;
2) genomic dna of preparation testing sample uses the primer in the step 1), treats survey sample gene group DNA and carries out pcr amplification, obtains target sequence;
3) markers step 2) in the target sequence that obtains;
4) with the nucleotide sequence gene chip hybridization shown in the target sequence behind the mark and the SEQ ID NO:1-8;
5) obtain hybridization signal and analyze results of hybridization with biochip scanner.
Wherein, the primer described in the step 1) comprises at least a of the nucleotide sequence shown in the SEQ ID NO:9-24, the oligonucleotide sequence of each bar primer probe by 5 ' end to 3 ' end form and corresponding amplification effect is:
P1(SEQ ID NO:9) TTTGATCCACCGACTTACAGCC is used for the upstream primer of amplification celery anaphylactogen MTD gene;
P2 (SEQ ID NO:10) ATAAAAACAGATAACGCTGACTCATCAC is used for the downstream primer of amplification celery anaphylactogen MTD gene;
P3 (SEQ ID NO:11) ATAACAGCGCAATCCTCTCCC is used for the upstream primer of amplification fishing line mitochondrial DNA;
P4 (SEQ ID NO:12) GCTGCACCATTAGGATGTCCT is used for the downstream primer of amplification fishing line mitochondrial DNA;
P5 (SEQ ID NO:13) GCAACAGGAGCAACAGTTCAAG is used for the upstream primer of peanut Arah1 gene;
P6 (SEQ ID NO:14) CGCTGTGGTGCCCTAAGG is used for the downstream primer of amplification peanut Arah1 gene;
P7 (SEQ ID NO:15) CAACAATTTTCTCAGCCCCAACA is used for the upstream primer of amplification wheat GAG56D gene;
P8 (SEQ ID NO:16) TTCTTGCATGGGTTCACCTGTT is used for the downstream primer of amplification wheat GAG56D gene;
P9 (SEQ ID NO:17) CCAGAGGGCTAGGGACCTTC is used for the upstream primer of amplification sesame Sal gene;
P10 (SEQ ID NO:18) CTCGGAATTGGCATTGCTG is used for the downstream primer of amplification sesame Sal gene;
P11 (SEQ ID NO:19) CTCTACCCTTGCTGAAAC is used for the upstream primer of amplification chicken Mitochondrial DNA gene
P12 (SEQ ID NO:20) TGATGAGGCTTACTGTGC is used for the downstream primer of amplification chicken Mitochondrial DNA gene
P13(SEQ ID NO:21) ACCTTGGTACTGGTGCTAC is used for the upstream primer of amplification soybean Lectin gene
P14 (SEQ ID NO:22) ACCTTGGCTACTTTATTGTT is used for the downstream primer of amplification soybean Lectin gene
P15 (SEQ ID NO:23) TTTGGTTGAAGGAGATGCTC is used for the upstream primer of amplification almond Pru dul gene
P16(SEQ ID NO:24) TAGTTGCTGGTGCTCTTTATG is used for the downstream primer of amplification almond Pru dul gene
Another object of the present invention provides a kind of test kit that detects anaphylactogen in the food, and this test kit comprises above-mentioned gene chip, and described gene chip comprises at least a of the nucleotide sequence of SEQ ID NO:1-8 or its complementary nucleotide sequence; The primer that also comprises pcr amplification, this primer has nucleotide sequence at least a of SEQ ID NO:9-24, wherein, be used for the PCR primer of amplification celery, chicken, fish, peanut, sesame, soybean, wheat, almond respectively according to MTD gene, mitochondrion DNA, plastosome 16s, Arah1 gene, Sal gene, Lectin gene, GAG56D gene, Pru dul gene design.
Test kit of the present invention also comprises hybridizing box, hybridization solution etc.
Test kit of the present invention can be used for the detection of common food allergens celery, chicken, fish, peanut, sesame, soybean, wheat, at least a pathogenic bacterium of almond.
Compared with prior art, the present invention adopts the beneficial effect of technique scheme to be:
Primer and the probe of the design of existing chip technology institute substantially all are positioned at 16s rRNA gene, the present invention will have on the anaphylactogen specific gene of obvious evolutionary edge, design specific probe and primer, effectively avoided resolving power lower, can't distinguish the drawback of planting, simultaneously, sensing range of the present invention has contained common food allergens in the daily life substantially, has greatly remedied the shortcoming of detection chip sensing range shortcoming in the prior art.
Detection sensitivity of the present invention is high, and the detection kit that provides and detection method susceptibility thereof are high, and accuracy of detection is high, and the dna profiling that can detect 1ng/ μ l is seen (Figure 11, Figure 12, Figure 13).Can carry out comprehensively, system, detect accurately and identify common pathogen.
Gene chip of the present invention can detect 8 kinds of common food allergens celeries, chicken, fish, peanut, sesame, soybean, wheat, almond specifically.This detection method approximately needs 24 hours.On a sheet base, can detect simultaneously 8 samples, reduce cost, realize high-throughput, detect simultaneously the purpose of a plurality of samples, be particularly suitable for detecting the sample of those Diversities.
As seen from the above technical solutions, the present invention introduces chip technology in the food allergen rapid detection, set up a kind of fast, sensitivity, high-throughput, accuracy is high, repeatability is strong brand-new food allergen detection gene chip and detection method thereof, utilize gene chip of the present invention can reach the purpose that 8 kinds of main food are detected, because easy and simple to handle, accuracy is high, can once finish other detection of multiple-type, repeatability is strong, therefore for the irritated original important using value of hospital's monitoring, realize the fast and accurately detection to food allergen.
Description of drawings
Fig. 1 is gene chip construction profile synoptic diagram of the present invention;
Fig. 2 is the single dot matrix structural representation of chip of the present invention;
Fig. 3 is for being the results of hybridization synoptic diagram that utilizes genechip detection celery anaphylactogen of the present invention;
Fig. 4 is for utilizing genechip detection flesh of fish anaphylactogen results of hybridization synoptic diagram of the present invention;
Fig. 5 is for utilizing genechip detection Peanut Allergen results of hybridization synoptic diagram of the present invention;
Fig. 6 is the results of hybridization synoptic diagram that utilizes genechip detection wheat of the present invention;
Fig. 7 is the results of hybridization synoptic diagram that utilizes genechip detection sesame of the present invention;
Fig. 8 is the results of hybridization synoptic diagram that utilizes genechip detection chicken anaphylactogen of the present invention;
Fig. 9 is the results of hybridization synoptic diagram that utilizes genechip detection original soybean sensitive of the present invention;
Figure 10 is the results of hybridization synoptic diagram that utilizes genechip detection almond anaphylactogen of the present invention;
Figure 11 is the results of hybridization synoptic diagram take soybean (1 ng/uL) as the example detection sensitivity;
The continuity of Figure 12 Figure 11 (10 ng/uL);
The continuity of Figure 13 Figure 11 (100 ng/uL).
Embodiment
For above and other objects of the present invention, feature and advantage can be become apparent, the below is especially exemplified by preferred embodiment, and the cooperation Figure of description, is described in detail below.The used all ingredients of the present invention all has commercially available.
Embodiment 1The design of probe and preparation
1. sequence obtains:
The MTD gene of celery, the mitochondrion DNA of chicken, the plastosome 16s of fish, the Arah1 gene of peanut, the Sal gene of sesame, soybean (bean sprouts) Lectin gene, the GAG56D gene of wheat, the Pru dul gene of almond are all downloaded from the GenBank public database and are obtained.
2. probe design is for example:
(1) the MTD gene probe of celery: the MTD gene gene order of celery is imported in the Glustal X software, chooses one and represent sequence and in common data NCBI, do the blastn comparison, definite could be as the position of special target spot and special target spot.Sequence is imported in the OligoArray2.0 software.Parameter setting is as follows :-n 20;-l 30;-L 40;-D 3000;-t 79;-T 90; 65 ℃ of-s; 65 ℃ of-x;-N 2;-p 33, and-P 65;-m GGGGG CCCCC TTTTT AAAAA;-g 15.The online designing probe of working procedure.
3. probe is synthetic: entrust probe Synesis Company (Beijing AudioCodes company) synthetic, for subsequent use after 5 ' of the probe sequence in the following table 1 is held N T of prolongation and amination.
4. probe screening: with making gene chip with gene chip sample applying instrument point at glass chip after synthetic probe dissolving and an amount of dilution, carry out the probe screening by hybrid experiment, finally obtain for the preparation of the required special probe of gene chip of the present invention.
The method of design of other probe is identical with celery MTD gene probe method of design, and the design variable of use is also identical.
The present invention carries out the probe screening by hybrid experiment repeatedly, and the preferred probe that obtains is as shown in table 1:
Table 1: the sequence oligonucleotide probe of selecting on the gene chip of the present invention and detectable food allergen
| SEQ ID | The probe numbering | Sequence (5'-3') | |
| NO:1 | NO.1 | ATTACATGCTGAGTCACGATGAGCGTG | For detection of the celery anaphylactogen; |
| NO:2 | NO.2 | TTTACGACCTCGATGTTGGATCAGGAC | For detection of flesh of fish anaphylactogen; |
| NO:3 | NO.3 | AGCTCAGGAACTTGCCTCAACAGTGCG | For detection of Peanut Allergen; |
| NO:4 | NO.4 | TTCCCGCAGCCCCAACAACCGCAACAA | Former for detection of Wheat Dood Allergy; |
| NO:5 | NO.5 | TCGCAGGTGCAACATGCGACCCCAGCA | For detection of the sesame anaphylactogen; |
| NO:6 | NO.6 | CCTTTCATCCTCATTTCTATGAATCCG | For detection of the chicken anaphylactogen; |
| NO:7 | NO.7 | CACAAAAAGGCTTGCAGATGGGCTTGC | For detection of original soybean sensitive; |
| NO:8 | NO.8 | GAGAAAATCAGTTATGAGATTAAGTT | For detection of the almond anaphylactogen; |
With reference to Fig. 1, be gene chip construction profile synoptic diagram of the present invention, the top of this gene chip is the point sample district, and the bottom is label area, and wherein regular distribution has dot matrix area in the point sample district.The lattice position of probe on glass chip is: the upper end of the first horizontally-arranged dot matrix area is 9.25mm apart from the top of glass chip, the left-hand point array area is 4.5mm apart from left side, the right-hand point array area of glass chip apart from the right side of glass chip, transverse distance between two dot matrix areas is 13.5mm with vertical distance, and the distance between the 3rd horizontally-arranged dot matrix area and the 4th horizontally-arranged dot matrix area is 13.5mm.
With reference to Fig. 2, be the single point array structure synoptic diagram of chip of the present invention, Cy3 wherein represents fluorescent probe, the nucleotide sequence of the numeral of numbering correspondence wherein is consistent with the nucleotide sequence shown in the table 1.
Embodiment 2The design of primer and preparation
1. design of primers is for example:
The MTD gene of celery, the mitochondrion DNA of chicken, the plastosome 16s of fish, the Arah1 gene of peanut, the Sal gene of sesame, soybean (bean sprouts) Lectin gene, the GAG56D gene of wheat, the Pru dul gene PCR amplimer of almond: with the MTD gene of celery, the mitochondrion DNA of chicken, the plastosome 16s of fish, the Arah1 gene of peanut, the Sal gene of sesame, soybean (bean sprouts) Lectin gene, the GAG56D gene of wheat, the Pru dul gene order of almond imports in the Glustal X software, therefrom chooses a representational sequence and imports in Primer Primier 5.0 softwares preseting length 70bp～10bp, G+C% value 40% ~ 60%, Hairpin:NONE, Dimer:NONE, False Priming:NONE, Cross Dimer:NONE.And seek out the nucleotide sequence district that is fit to design of primers, and its characteristics meet the following conditions substantially: 1, and this zone should include the variable region that is easy to the specific probe design, guarantees that the difference of Nucleotide between probe is greater than more than 4; 2, these both sides, zone are the design that constant region can satisfy primer; 3, designed primer extension product is unsuitable excessive, otherwise affects the susceptibility of PCR.
2. primer is synthetic: the primer sequence in the table 2 is entrusted probe Synesis Company (handsome biotech company) synthetic (PAGE purifying), and for subsequent use.
Table 2 is used for the pcr amplification primer sequence that the health check-up of tap water encountered pathogenic is surveyed
| Numbering | SEQ ID | Sequence (5 '-3 ') | The primer effect |
| P1 | NO:9 | TTTGATCCACCGACTTACAGCC | The upstream primer that is used for amplification celery anaphylactogen MTD gene |
| P2 | NO:10 | ATAAAAACAGATAACGCTGACTCATCAC | The downstream primer that is used for amplification celery anaphylactogen MTD gene |
| P3 | NO:11 | ATAACAGCGCAATCCTCTCCC | The upstream primer that is used for amplification amplification fishing line mitochondrial DNA |
| P4 | NO:12 | GCTGCACCATTAGGATGTCCT | The downstream primer that is used for amplification fishing line mitochondrial DNA |
| P5 | NO:13 | GCAACAGGAGCAACAGTTCAAG | The upstream primer that is used for peanut Arah1 gene |
| P6 | NO:14 | CGCTGTGGTGCCCTAAGG | The downstream primer that is used for amplification peanut Arah1 gene |
| P7 | NO:15 | CAACAATTTTCTCAGCCCCAACA | The upstream primer that is used for amplification wheat GAG56D gene |
| P8 | NO:16 | TTCTTGCATGGGTTCACCTGTT | The downstream primer that is used for amplification wheat GAG56D gene |
| P9 | NO:17 | CCAGAGGGCTAGGGACCTTC | The upstream primer that is used for amplification sesame Sal gene |
| P10 | NO:18 | CTCGGAATTGGCATTGCTG | The downstream primer that is used for amplification sesame Sal gene |
| P11 | NO:19 | CTCTACCCTTGCTGAAAC | The upstream primer that is used for amplification chicken Mitochondrial DNA gene |
| P12 | NO:20 | TGATGAGGCTTACTGTGC | The downstream primer that is used for amplification chicken Mitochondrial DNA gene |
| P13 | NO:21 | ACCTTGGTACTGGTGCTAC | The upstream primer that is used for amplification soybean Lectin gene |
| P14 | NO:22 | ACCTTGGCTACTTTATTGTT | The downstream primer that is used for amplification soybean Lectin gene |
| P15 | NO:23 | TTTGGTTGAAGGAGATGCTC | The upstream primer that is used for amplification almond Pru dul gene |
| P16 | NO:24 | TAGTTGCTGGTGCTCTTTATG | The downstream primer that is used for amplification almond Pru dul gene |
Illustrate:
(1) P1/P2 is used for amplification and is used for amplification celery anaphylactogen MTD gene.
(2) P3/P4 is used for amplification and is used for amplification fishing line mitochondrial DNA gene.
(3) P5/P6 is used for amplification and is used for peanut Arah1 gene.
(4) P7/P8 is used for amplification wheat GAG56D gene.
(5) P9/P10 is used for amplification sesame Sal gene.
(6) P11/P12 is used for amplification chicken Mitochondrial DNA gene.
(7) P13/P14 is used for amplification soybean Lectin gene.
(8) P15/P16 is used for amplification almond Pru dul gene.
Embodiment 3Utilize the preparation of gene chip rapid detection common food anaphylactogen and test kit
1. sample preparation:
(1) buys the required food of experiment in food market, Tianjin Binhai development area
(2) extract genomic test kit step according to the precious biotech firm in Dalian and extract genome.
(3) return the lysin gene group, can for detection of or-20 ℃ of preservations.
Attached: the test kit title: the DNA Extraction Kit for GMO Detection Ver.2.0 of Bao Bio-Engineering Company)
The precious TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.4.0 of biotech firm)
2. amplified target sequence: get the 3ul supernatant that said gene group extracting method extracts and add in the PCR reaction mixture as template, PCR reaction mixture prescription is as shown in table 3 below.(annotate: the PCR damping fluid in the following table 3-table 4, MgCl2, dNTP mixture, Taq enzyme are all available from Sangon company)
Table 3 PCR reaction mixture prescription
Constituent concentration application of sample amount (μ l)
ddH
2O - 11.7
10 * PCR damping fluid 10 * 2.5
MgCl
2 25mM 2.5
DNTP mixture 10mM 0.5
Primer mixture I note
4.5
Taq enzyme 5U/ μ l 0.3
Cumulative volume 25
Annotate:
The concentration of each primer is in the primer mixture I: P1-P8 respectively is 3.33 μ molL
-1P3, P4 are 1.11 μ molL
-1P5, P6 are 0.28 μ molL
-1;P7, P8 are 0.56 μ molL
-1
Reaction tubes is put into PCR instrument (Biometra), and the loop parameter of setting is as follows:
80 ℃ 10 minutes
94 ℃ 5 minutes
94 ℃ 30 seconds
52 ℃ 1 minute 15 seconds
Got back to for the 3rd step totally 35 circulations in 1 minute for 72 ℃
72 ℃ 5 minutes
Celery (MTD gene) amplification size is 151bp, the mitochondrion DNA cloning size of chicken is 671bp, the plastosome 16s amplification size of fish is 86bp, the Arah1 gene amplification size of peanut is 72bp, the Sal gene amplification size of sesame is 62bp, soybean (bean sprouts) Lectin gene amplification size is 530bp, and the GAG56D gene amplification size of wheat is 122bp, and the Pru dul gene amplification size of almond is 108bp.
3. fluorescent mark target sequence: get 10 μ l amplified productions, add in the mark mixed solution, the labeled reactant mixture formula is as shown in table 4 below.
Table 4 labeled reactant mixture formula
Constituent concentration application of sample amount (μ l)
ddH
2O - 6.65
10 * PCR damping fluid 10 * 2.5
MgCl
2 25mM 2.5
DNTP mixture 10mM 0.5
Primer mixture II note
2.25
Fluorescein Cy3-dUTP 25nM 0.3
Taq enzyme 5U/ μ l 0.3
Cumulative volume 25
Annotate:
Contained each primer concentration is in the primer mixture II: P2 is 3.33 μ molL
-1P4 is 1.11 μ molL
-1P6 is 0.28 μ molL
-1P8 is 0.56 μ molL
-1
Reaction tubes is put into PCR instrument (Biometra), and the loop parameter of setting is as follows:
80 ℃ 10 minutes
94 ℃ 5 minutes
94 ℃ 30 seconds
52 ℃ 1 minute 15 seconds
Got back to for the 3rd step totally 35 circulations in 1 minute for 72 ℃
72 ℃ 5 minutes
4. hybridization: place 65 ℃ of baking ovens to dry marked product, in hybridizing box (Bo Ao company), add in advance 100 μ l ddH2O to keep humidity.Getting 18 μ l-2 hybridization solutions mixes with the oven dry marked product, and the probe array that is added in the tap water encountered pathogenic microorganism detection gene chip of preparation among the embodiment one is regional, cover cover plate (Bo Ao company product, production number 430042) (notes between cover plate and the slide glass bubble being arranged), cover tightly hybridizing box, hybridization is 12 hours in 44 ℃ of water-baths.
Hybridization solution prescription: 25% formamide, 0.1% SDS, 6 * SSPE.
5. washing: when hybridizing to, take out hybridizing box, remove cover plate, gene chip was washed 3 minutes in washing lotion A successively, washing is 3 minutes among the washing lotion B, and washing is 90 seconds among the washing lotion C, and is air-dry in the air.
Washing lotion A:1 * SSC (sodium-chlor-sodium citrate solution); 0.1% SDS
Washing lotion B:0.05 * SSC
Washing lotion C:95% ethanol
6. scanning: with GenePix personal 4100A biochip scanner (AXON instrument) scanning, used parameter is as follows:
Software and version: GenePix Pro 6.0
official name: 575DF35
PMT Gain：550
Scanning resolution: 10 μ m
Scanning result saves as JPG, TIF, GPR form
Hybridization scanning result when detecting respectively the detection of common food allergens involved in the present invention with gene chip of the present invention is respectively shown in Fig. 3-13.
Coming as can be seen from Figure 3 the probe NO.1 of celery is special to celery, can detect with this test kit.
The probe NO.2 that oppresses as can be seen from Figure 4 is special to the flesh of fish, can detect with this test kit.
Coming as can be seen from Figure 5 the probe NO.3 of peanut is special to peanut, can detect with this test kit.
Coming as can be seen from Figure 6 the probe NO.4 of celery is special to wheat, can detect with this test kit.
Coming as can be seen from Figure 7 the probe NO.5 of celery is special to sesame, can detect with this test kit.
Coming as can be seen from Figure 8 the probe NO.6 of celery is special to chicken, can detect with this test kit.
Coming as can be seen from Figure 9 the probe NO.7 of celery is special to soybean, can detect with this test kit.
Coming as can be seen from Figure 10 the probe NO.8 of celery is special to almond, can detect with this test kit.
Can find out from Figure 11,12,13, this test kit can also detect at 1 ng/uL, illustrates that sensitivity is very high.
7. the analysis interpretation of results of hybridization: this chip is low density chip, and number of probes is less, and detected result can be judged by naked eyes.According to the hybridization image that scans, as image coordinate, judge the position of the specific probe that fluorescent signal occurs with the position of fluorescent probe, contrast dot matrix layout viewing is judged pathogenic agent.
8. the hybridization kit that utilizes above-mentioned experimental procedure to obtain can be used for detecting common food allergens celery, chicken, fish, peanut, sesame, soybean, wheat, at least a detection of almond, and wherein this test kit is also with the specification sheets that uses this test kit etc.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment does.
SEQUENCE LISTING
＜110〉Tianjin Biochip Technology Co., Ltd
＜120〉gene chip and the detection kit of the common food allergens of detection
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> DNA
＜213〉the MTD gene of celery
<400> 1
attacatgct gagtcacgat gagcgtg 27
<210> 2
<211> 27
<212> DNA
＜213〉Mitochondrial DNA of fish
<400> 2
tttacgacct cgatgttgga tcaggac 27
<210> 3
<211> 27
<212> DNA
＜213〉the Arah1 gene of peanut
<400> 3
agctcaggaa cttgcctcaa cagtgcg 27
<210> 4
<211> 27
<212> DNA
＜213〉the GAG56D gene of wheat
<400> 4
ttcccgcagc cccaacaacc gcaacaa 27
<210> 5
<211> 27
<212> DNA
＜213〉the Sal gene of sesame
<400> 5
tcgcaggtgc aacatgcgac cccagca 27
<210> 6
<211> 27
<212> DNA
＜213〉Mitochondrial DNA of chicken
<400> 6
cctttcatcc tcatttctat gaatccg 27
<210> 7
<211> 27
<212> DNA
＜213〉soybean (bean sprouts) Lectin gene
<400> 7
cacaaaaagg cttgcagatg ggcttgc 27
<210> 8
<211> 26
<212> DNA
＜213〉the Pru dul gene of almond
<400> 8
gagaaaatca gttatgagat taagtt 26
<210> 9
<211> 22
<212> DNA
＜213〉MTD gene
<400> 9
tttgatccac cgacttacag cc 22
<210> 10
<211> 28
<212> DNA
＜213〉MTD gene
<400> 10
ataaaaacag ataacgctga ctcatcac 28
<210> 11
<211> 21
<212> DNA
＜213〉fishing line mitochondrial DNA
<400> 11
ataacagcgc aatcctctcc c 21
<210> 12
<211> 21
<212> DNA
＜213〉fishing line mitochondrial DNA
<400> 12
gctgcaccat taggatgtcc t 21
<210> 13
<211> 22
<212> DNA
＜213〉peanut Arah1 gene
<400> 13
gcaacaggag caacagttca ag 22
<210> 14
<211> 18
<212> DNA
＜213〉peanut Arah1 gene
<400> 14
cgctgtggtg ccctaagg 18
<210> 15
<211> 23
<212> DNA
＜213〉wheat GAG56D gene
<400> 15
caacaatttt ctcagcccca aca 23
<210> 16
<211> 22
<212> DNA
＜213〉wheat GAG56D gene
<400> 16
ttcttgcatg ggttcacctg tt 22
<210> 17
<211> 20
<212> DNA
＜213〉sesame Sal gene
<400> 17
ccagagggct agggaccttc 20
<210> 18
<211> 19
<212> DNA
＜213〉sesame Sal gene
<400> 18
ctcggaattg gcattgctg 19
<210> 19
<211> 18
<212> DNA
＜213〉Mitochondrial DNA gene
<400> 19
ctctaccctt gctgaaac 18
<210> 20
<211> 18
<212> DNA
＜213〉chicken Mitochondrial DNA gene
<400> 20
ctctaccctt gctgaaac 18
<210> 21
<211> 19
<212> DNA
＜213〉soybean Lectin gene
<400> 21
accttggtac tggtgctac 19
<210> 22
<211> 20
<212> DNA
＜213〉soybean Lectin gene
<400> 22
accttggcta ctttattgtt 20
<210> 23
<211> 20
<212> DNA
＜213〉almond Pru dul gene
<400> 23
tttggttgaa ggagatgctc 20
<210> 24
<211> 21
<212> DNA
＜213〉almond Pru dul gene
<400> 24
tagttgctgg tgctctttat g 21
Claims (10)
1. gene chip that detects anaphylactogen in the common food comprises solid phase carrier and is fixed on oligonucleotide probe on this solid phase carrier, it is characterized in that this oligonucleotide probe comprises at least a in the following dna fragmentation:
1. celery MTD gene
2. chicken Mitochondrial DNA
3. the plastosome 16SDNA of fish
4. the Arah1 gene of peanut
5. the Sal gene of sesame
6. the Lectin gene of soybean
7. the GAG56D gene of wheat
8. the Pru dul gene of almond
9. 1. the dna fragmentation of choosing ~ 8..
2. gene chip according to claim 1, it is characterized in that described oligonucleotide probe has the nucleotide sequence shown in the SEQ ID NO:1-8, or be different from SEQ ID NO:1-8 but the coded identical nucleotide sequence of aminoacid sequence of the identical nucleotide sequence of the coded aminoacid sequence of aminoacid sequence and the SEQ ID NO:1-8 of coding and SEQ ID NO:1-8.
3. gene chip claimed in claim 1 detects the application aspect the anaphylactogen test kit in common food celery, chicken, fish, peanut, sesame, soybean, wheat, the almond in preparation.
4. the preparation method of the described gene chip of claim 1 is characterized in that being undertaken by following step:
1) specific gene of common food allergens according to claim 1 designs and prepares the primer for pcr amplification;
2) genomic dna of preparation testing sample uses the primer in the step 1), treats survey sample gene group DNA and carries out pcr amplification, obtains target sequence;
3) markers step 2) in the target sequence that obtains;
4) with the target sequence behind the mark and gene chip hybridization claimed in claim 1;
5) obtain hybridization signal and analyze results of hybridization with biochip scanner.
5. preparation method according to claim 4 is characterized in that the primer described in the step 1) comprises at least a of the nucleotide sequence shown in the SEQ ID NO:9-24.
6. a test kit that detects common anaphylactogen in the food is characterized in that comprising gene chip claimed in claim 1.
7. test kit according to claim 6 is characterized in that described gene chip comprises at least a of the nucleotide sequence shown in the SEQ ID NO:1-8 or its complementary nucleotide sequence.
8. according to claim 6 or 7 described test kits, it is characterized in that also comprising the primer of pcr amplification, this primer has nucleotide sequence at least a of SEQ ID NO:9-24.
9. test kit according to claim 6 is characterized in that also comprising hybridizing box, hybridization solution.
10. test kit claimed in claim 6 detects the application aspect the anaphylactogen in common food celery, chicken, fish, peanut, sesame, soybean, wheat, the almond in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100192543A CN103060462A (en) | 2013-01-18 | 2013-01-18 | Gene chip and detection kit for detecting common food allergens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013100192543A CN103060462A (en) | 2013-01-18 | 2013-01-18 | Gene chip and detection kit for detecting common food allergens |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103060462A true CN103060462A (en) | 2013-04-24 |
Family
ID=48103353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013100192543A Pending CN103060462A (en) | 2013-01-18 | 2013-01-18 | Gene chip and detection kit for detecting common food allergens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103060462A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484456A (en) * | 2013-09-23 | 2014-01-01 | 曹际娟 | Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer |
| CN104561256A (en) * | 2014-05-29 | 2015-04-29 | 天津出入境检验检疫局动植物与食品检测中心 | Walnut mass spectrometric detection characteristic sequence group and detection kit |
| CN104569243A (en) * | 2014-05-29 | 2015-04-29 | 天津出入境检验检疫局动植物与食品检测中心 | Peanut mass spectrometric detection signature sequence group and detection kit |
| CN110396554A (en) * | 2019-05-07 | 2019-11-01 | 广东出入境检验检疫局检验检疫技术中心 | Utilize the method for dual digital pcr quantitative detection Li Rongzhong mung bean ingredient |
| CN113430258A (en) * | 2021-07-09 | 2021-09-24 | 湖北省食品质量安全监督检验研究院 | Polynucleotide pair and probe combination for detecting allergen components and application thereof |
| CN113755555A (en) * | 2021-09-03 | 2021-12-07 | 浙江工商大学 | Capture probe set for detecting food allergen, preparation method and application thereof |
| CN114214452A (en) * | 2021-12-31 | 2022-03-22 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | Chip kit for detecting plant-derived allergen components |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899498A (en) * | 2009-05-15 | 2010-12-01 | 天津出入境检验检疫局动植物与食品检测中心 | Standard substance for detecting allergen almond components in foods |
| CN102108404A (en) * | 2010-12-08 | 2011-06-29 | 中国检验检疫科学研究院 | Visible chip-based method for detecting food allergens |
-
2013
- 2013-01-18 CN CN2013100192543A patent/CN103060462A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899498A (en) * | 2009-05-15 | 2010-12-01 | 天津出入境检验检疫局动植物与食品检测中心 | Standard substance for detecting allergen almond components in foods |
| CN102108404A (en) * | 2010-12-08 | 2011-06-29 | 中国检验检疫科学研究院 | Visible chip-based method for detecting food allergens |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484456A (en) * | 2013-09-23 | 2014-01-01 | 曹际娟 | Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer |
| CN103484456B (en) * | 2013-09-23 | 2015-04-15 | 曹际娟 | Loop-mediated isothermal amplification primer and kit for detecting apium graveolens ingredients and application of loop-mediated isothermal amplification primer |
| CN104561256A (en) * | 2014-05-29 | 2015-04-29 | 天津出入境检验检疫局动植物与食品检测中心 | Walnut mass spectrometric detection characteristic sequence group and detection kit |
| CN104569243A (en) * | 2014-05-29 | 2015-04-29 | 天津出入境检验检疫局动植物与食品检测中心 | Peanut mass spectrometric detection signature sequence group and detection kit |
| CN110396554A (en) * | 2019-05-07 | 2019-11-01 | 广东出入境检验检疫局检验检疫技术中心 | Utilize the method for dual digital pcr quantitative detection Li Rongzhong mung bean ingredient |
| CN113430258A (en) * | 2021-07-09 | 2021-09-24 | 湖北省食品质量安全监督检验研究院 | Polynucleotide pair and probe combination for detecting allergen components and application thereof |
| CN113430258B (en) * | 2021-07-09 | 2022-09-20 | 湖北省食品质量安全监督检验研究院 | Polynucleotide pair and probe combination for detecting allergen components and application thereof |
| CN113755555A (en) * | 2021-09-03 | 2021-12-07 | 浙江工商大学 | Capture probe set for detecting food allergen, preparation method and application thereof |
| CN114214452A (en) * | 2021-12-31 | 2022-03-22 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | Chip kit for detecting plant-derived allergen components |
| CN114214452B (en) * | 2021-12-31 | 2024-04-05 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | Chip kit for detecting plant-derived allergen components |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103060462A (en) | Gene chip and detection kit for detecting common food allergens | |
| US20210109076A1 (en) | Systems and methods for allergen detection | |
| AU2017272280A1 (en) | Allergen detection | |
| CN109439767A (en) | Dual real-time fluorescence PCR primer sets, kit and method a kind of while that identify atlantic salmon and rainbow trout | |
| Sato et al. | Patterns of trematode infections of Opisthorchis viverrini (Opisthorchiidae) and Haplorchis taichui (Heterophyidae) in human populations from two villages in Savannakhet Province, Lao PDR | |
| Kareshk et al. | Molecular detection and genetic diversity of Toxoplasma gondii in different tissues of sheep and goat in Eastern Iran | |
| KR101219763B1 (en) | Development of pcr primers for species identification of fishes | |
| CN109554449A (en) | A kind of multiple PCR method that can detect 7 virulence genes of Aeromonas simultaneously | |
| CN106811514B (en) | Specific real-time fluorescence detection method for biological components in Amydae and kit thereof | |
| CN101851669B (en) | Real-time fluorescent PCR quantitative detection method of original soybean sensitive components in food | |
| CN110066880A (en) | A kind of method of four large Chinese carp fish product of fast qualitative detection | |
| CN101381767A (en) | Universal real time fluorescent PCR detection method of trichinella | |
| Martínez-García et al. | OsHV-1 and notifiable protozoa in healthy Crassostrea corteziensis cultured in two distant areas of the Gulf of California | |
| CN103114142B (en) | Real-time polymerase chain reaction (PCR) detection primer, kit and detection method for detecting puffer fish ingredients | |
| KR102117391B1 (en) | Multiplex pcr primers for detection of shellfishes causing allergy, and uses thereof | |
| CN101812509B (en) | PCR identification method for equine derived material in traditional Chinese medicament | |
| KR101277798B1 (en) | Development of pcr primers for species identification of meat | |
| KR101277799B1 (en) | Development of pcr primers for species identification of poultry | |
| KR101277801B1 (en) | Development of pcr primers for species identification of root vegetables | |
| CN102766686B (en) | Preparation and detection method of rapid detection kit for garlic component in food and beverage | |
| CN101748216B (en) | Real time fluorescent PCR detecting primer and probe for food sensibiligen shrimp component, kit and detecting method | |
| CN108611442A (en) | The fluorescence quantitative RT-PCR primer and probe and method of a kind of detection pig atypia pestivirus and application | |
| JP2016187329A (en) | Primer, and method for detecting tree nut | |
| Fujimura et al. | Specific discrimination of chicken DNA from other poultry DNA in processed foods using the polymerase chain reaction | |
| CN103667538A (en) | Gene chip, kit and method for detecting three viruses for immunosuppression disease of chickens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130424 |