CN103160608A - Food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method - Google Patents
Food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method Download PDFInfo
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Abstract
The invention relates to a food allergen lupin component LAMP (loop-mediated isothermal amplification) field quick detection method. The invention discloses a food allergen lupin component LAMP detection method for the first time. The invention uses lupin ITS1 gene as a target gene for identification, and designs LAMP amplification primers with favorable specificity. The method provided by the invention can be well used for identifying the food allergen lupin component, and has favorable repeatability and sensitivity.
Description
Technical field
The present invention relates to molecular biology and nucleic acid detection technique field.Particularly, the present invention relates to LAMP detection method and the test kit of food allergens lupine composition in food.
Background technology
Food anaphylaxis is a kind of untoward reaction that people produce food, belongs to a kind of transformation reactions that body produces allogenic material, and at present, the internationally recognized main eight irritated food of large class are: soybean, peanut, wheat, egg, milk, fish, nut, crustaceans.The general symptom of food anaphylaxis comprises vomiting, stomachache, diarrhoea etc., also comprises skin reaction and respiratory symptom, even general anaphylaxis shock or death can occur in serious situation.Treatment there is no effect method for food anaphylaxis, and strictly avoiding the edible food that contains the anaphylactogen composition is the most effective therapy.Food prohibited is difficult to realize completely, because food allergen may be hidden in the middle of other food.For protecting irritated crowd, the law bill of anaphylactogen sign in standard food is revised and put into effect in national governments and international organization in succession, and the mark of anaphylactogen composition on food labelling is made strict regulation.The states such as the U.S., European Union, Canada require the Main Foods anaphylactogen kinds such as mandatory sign soybean, wheat.
Lupine (Lupinus polyphyllus), pulse family Papillionoideae lupinus, annual or per nnial herb can be divided into according to purposes and view and admire lupine and edible lupine.Edible lupin protein matter content is up to 50%, oleaginousness is 5-20%, now, farming species articles for use kind mainly contains spends lupine, lupinus augustifolius, chrysanthemum lupine and pearl lupine in vain, and there is plantation widely in the many countries in Australia and Europe, Asia and Africa.Due to its special functional and nutrition feature, what lupine was more applies in food-processing, and on the one hand, lupine can replace soybean and be applied in food in addition, has so just reduced the risk that in food, genetically engineered soybean exists.But the lupine sensitization has brought puzzlement, the mandatory sign anaphylactogen of European Union's laws and regulations requirement lupine composition for a lot of sensitive individuals.Lupine allergy usually occurs and peanut allergy cross reaction and causing, very likely also irritated to lupine to the patient of peanut allergy, its allergic symptom main manifestations is contact rubella and asthma.Therefore particularly important to the detection of lupine composition.
Detection method about food ingredient has electrophoresis, chromatogram, immunochemistry, DNA technique etc. at present.Detection research for food allergy ultimate constituent in food, mainly contain at present take protein as the basis method (as the ELISA method) and take gene as the basis method (as PCR method), these two kinds of methods all need high-end plant and instrument, professional operator.This area is not also detected reagent and the method for the specific detection food allergens lupine composition respond well and simple to operate, that equipment requirements is low at present.
Summary of the invention
The object of the present invention is to provide food allergens lupine composition LAMP field fast detection method.
In a first aspect of the present invention, a kind of method of identifying food allergens lupine composition is provided, described method comprises:
Take the DNA of testing sample as template, increase with the primer of specific amplification lupine ITS1 gene; If the generation specific amplification shows to comprise food allergens lupine composition in testing sample.
In a preference, described amplification is ring mediated isothermal amplification (LAMP).
In another preference, the primer of described specific amplification lupine ITS1 gene comprises: upstream outer primer SEQ ID NO:1, downstream outer primer SEQ ID NO:2, upstream inner primer EQ ID NO:3, downstream inner primer SEQ ID NO:4.
In another preference, described ring mediated isothermal amplification comprises: 63 ± 1 ℃ of (preferably 63 ± 0.5 ℃) isothermal reaction 60 ± 10min (preferably 60 ± 5min), then 80 ± 2 ℃ of (preferably 80 ± 1 ℃) deactivation 5 ± 1min (preferably 5 ± 0.5min), finish reaction.
In another preference, the system of described ring mediated isothermal amplification comprises primer, Mg
2+, trimethyl-glycine, dNTPs; Wherein, Mg
2+Concentration 6 ± 1mM; Trimethyl-glycine concentration 0.8 ± 0.1M; DNTPs concentration 1.6 ± 0.2mM.
In another preference, after loop-mediated isothermal amplification finishes, add SYBR Green I fluorescence dye to observe colour-change in amplified production, do not have the negative tube of amplified production to be orange, the positive pipe of amplified production is arranged for green
In another preference, described testing sample is food or feed.
In another aspect of this invention, provide a kind of primer, described primer is the LAMP amplimer, comprising: upstream outer primer SEQ ID NO:1, downstream outer primer SEQ ID NO:2, upstream inner primer EQ ID NO:3, downstream inner primer SEQ ID NO:4.
In another aspect of this invention, provide the purposes of described primer, be used for identifying food allergens lupine composition from testing sample.
In another aspect of this invention, provide a kind of test kit of identifying food allergens lupine composition, comprising described primer.
In a preference, also comprise in described test kit:
The examination criteria product that contain the lupine composition;
DNA extraction reagent;
PH buffer reagent (as Tris-HCl (PH8.8));
Potassium ion solution;
Magnesium ion solution;
Ammonium ion solution;
Tween20；
Trimethyl-glycine;
dNTP；
Bst large fragment DNA polysaccharase;
Fluorescence dye (as SYBR Green I fluorescence dye); And/or
The working instructions of the method for identifying food allergens lupine composition are described.
Other side of the present invention due to the disclosure of this paper, is apparent to those skilled in the art.
Description of drawings
Fig. 1, food allergens lupine LAMP detect the real-time turbidity amplification collection of illustrative plates in primer reaction times.Wherein, CH1-3: negative control; CH4-6: positive control.
Fig. 2, food allergens lupine LAMP detect the real-time turbidity amplification collection of illustrative plates of primer system optimization.Wherein, CH1-2: negative control, CH3-4: positive control.
The real-time turbidity collection of illustrative plates of Fig. 3, food allergens lupine composition LAMP specific detection.
Wherein, A:CH1-8 is followed successively by chrysanthemum lupine, soybean, garbanzo, French beans, string bean, pea, Semen Phaseoli Vulgaris, mung bean; B:CH1-8 is followed successively by cowpea, broad bean, rice, corn, oat, wheat, buckwheat, peanut; C:CH1-8 is followed successively by almond, walnut, celery, Radix Dauci Sativae, potato, tomato, pears, orange; D:CH1-8 is followed successively by grapefruit, milk, egg, goat milk, duck's egg, goose egg, pigeon egg, pork; D:CH1-6 is followed successively by and spends lupine, grass shrimp, crab, salmon, negative control, negative control in vain.
The development process detected result of Fig. 4, food allergens lupine composition LAMP specific detection.
Wherein, 1-37 is followed successively by: chrysanthemum lupine, negative control, spend lupine, soybean, garbanzo, French beans, string bean, pea, Semen Phaseoli Vulgaris, mung bean, cowpea, broad bean, rice, corn, oat, wheat, buckwheat, peanut, almond, walnut, celery, Radix Dauci Sativae, potato, tomato, pears, orange, grapefruit, milk, goat milk, egg, duck's egg, goose egg, pigeon egg, pork, grass shrimp, crab, salmon in vain.
The real-time turbidity collection of illustrative plates that Fig. 5, food allergens lupine composition LAMP sensitivity detect.
Wherein, CH1:1% lupine; The CH2:0.1% lupine; The CH3:0.05% lupine; The CH4:0.01% lupine; The CH5:0.005% lupine; The CH6:0.001% lupine; The CH7:0.0005% lupine; CH8: negative control.
The development process detected result that Fig. 6, food allergens lupine composition LAMP sensitivity detect.
Wherein, 1-3:0% lupine; The 4-6:0.0005% lupine, 7-9:0.001% lupine, 10-12:0.1% lupine; 13: positive control; 14: negative control.
The real-time turbidity Detection of Stability figure that Fig. 7,0.1% lupine LAMP detect.
Wherein, A:CH1: positive control, CH2: negative control (ultrapure water), CH3-8:0.1% lupine analog sample; B:CH1-8 is 0.1% lupine analog sample; C:CH1-6 is 0.1% lupine analog sample.
The development process Detection of Stability figure that Fig. 8,0.1% lupine LAMP detect.
Wherein, 1: positive control, 2: negative control, 3-22:0.1% analog sample.
The real-time turbidity Detection of Stability figure that Fig. 9,0.001% lupine LAMP detect.
Wherein, A:CH1: positive control, CH2: negative control, CH3-6:0.001% lupine sample; B:CH1-8 is 0.001% lupine sample; C:CH1-8 is 0.001% lupine sample.
The development process Detection of Stability figure that Figure 10,0.001% lupine LAMP detect.
Wherein, 1: positive control, 2: negative control, 3-22:0.001% analog sample.
Embodiment
The inventor discloses a kind of ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) detection method of lupine anaphylactogen composition first through extensive and deep research and test.Through relatively screening, find that specificity is good with the target gene of lupine ITS1 gene as evaluation; And having designed the LAMP amplimer based on this, specific amplification (acquisition positive findings) can occur for the DNA that contains the lupine composition in described primer, and specific amplification (acquisition negative findings) is not occured the DNA that there is no the lupine composition.Method of the present invention can be applied to identify food allergens lupine composition well, and has good reproducibility, sensitivity.
Food in the market is that mixing element is made mostly, and various compositions are difficult to identification after fining-off.And other beans in leguminous plants beyond lupine and lupine proterties approach, increased especially the differentiation difficulty, even adopt the technology on some genes or protein level, also because some beans kinds are nearer on sibship, be difficult to find the lupine standard compliant, that detection accuracy is high, practical to detect target.For this reason, the inventor has found suitable detection target through deep research and a large amount of screenings, has developed the method for LAMP detection food allergens lupine composition based on this.
As used herein, described " lupine composition " refers to that specificity comes from the composition of lupine, such as feather fan bean powder etc.
As used herein, described " food " has comprised beverage.
The inventor is by the screening to primer, but the primer of acquisition one class specificity identification food allergens lupine composition, and specific amplification occurs in its DNA for lupine, and specific amplification is not occured the DNA that there is no the lupine composition.
Therefore, the invention provides a kind of primer, described primer is the LAMP amplimer, comprising: upstream outer primer SEQ ID NO:1, downstream outer primer SEQ ID NO:2, upstream inner primer EQ ID NO:3, downstream inner primer SEQ ID NO:4.
These primers of the present invention can also carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substances.
Utilize primer of the present invention, carry out the LAMP reaction, and detect or the observation that develops the color by turbidimeter, just can judge accurately and rapidly whether testing sample contains the lupine composition, and required sample size seldom.
The present invention also provides a kind of method of identifying food allergens lupine composition, and described method comprises: take the DNA of testing sample as template, increase with the primer of specific amplification lupine ITS1 gene; If the generation specific amplification shows to comprise food allergens lupine composition in testing sample.More preferably, based on the Auele Specific Primer that is applicable to identify food allergens lupine composition provided by the present invention, described method comprises: take the DNA of testing sample as template, increase with the primer shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4; If the generation specific amplification shows to comprise the lupine composition in testing sample.
LAMP is a kind of novelty of development in recent years and more ripe isothermal amplification.LAMP is the 4 kinds of special primers of 6 zone design for target gene, utilizes the strand displacement archaeal dna polymerase under isothermal condition, i.e. achievable nucleic acid amplification reaction.Compare with PCR method, LAMP does not need thermal cycler (PCR instrument), and owing to producing a large amount of by products-white magnesium pyrophosphate precipitation in the LAMP reaction, amplified production gets final product result of determination by visual inspection or turbidometer; Therefore LAMP is suitable for the scene or rapid detection is carried out in condition relatively poor laboratory.The LAMP technology has the advantages such as quick, accurate and easy and simple to handle.
The method of obtaining the DNA of testing sample is technology well-known to those skilled in the art, for example can take traditional phenol/chloroform/primary isoamyl alcohol method, the DNA extraction test kit that perhaps can adopt some to be purchased, and this class test kit is well known to those skilled in the art.
The invention still further relates to a kind of test kit for the identification of food allergens lupine composition, contain the LAMP amplimer for lupine ITS1 gene in described test kit.
In addition, described test kit also can contain other reagent of identifying the lupine composition, as (but being not limited to): the examination criteria product that contain the lupine composition; DNA extraction reagent; PH buffer reagent (as Tris-HCl (PH8.8)); Potassium ion solution; Magnesium ion solution; Ammonium ion solution; Tween20; Trimethyl-glycine; DNTP; Bst large fragment DNA polysaccharase; Fluorescence dye (as SYBR Green I fluorescence dye).
In addition, also can contain working instructions and/or the Standard operation procedure SOP of identifying food allergens lupine composition in described test kit.
Test kit of the present invention can be realized the purpose of rapid detection, batch detection food allergens lupine composition.
Major advantage of the present invention is:
(1) but disclose first a kind of primer of specificity identification food allergens lupine composition, described primer specificity is good, can realize specific amplification for the composition in lupine source, and can not specific amplification for other composition beyond lupine.And described primer has good reproducibility, result is reliable and stable.
(2) utilize described primer or contain the detection kit of described primer, can detect fast, in large quantity the lupine composition, distinguish rapidly and accurately the lupine composition from testing sample, and required sample size is few, simple to operate.
(3) method of the present invention is used for the detection at food safety accident scene and the sample detection that the food enterprises production process is controlled, and fills up the blank of food allergens field fast detection method.Applying ensureing the quality of product of method of the present invention, Protection of consumer right to know and preference safeguard that normal economic order etc. provides technical support.For market surveillance department and the inspection and quarantine department of food provides technical support.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Pehanorm Brooker etc. usually, molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that all specialties and scientific words and the one skilled in the art who uses in literary composition is familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
1, materials and methods
1.1 material
The chrysanthemum lupine, spend 36 kinds of materials such as lupine, soybean, garbanzo, French beans, string bean, pea, Semen Phaseoli Vulgaris, mung bean, cowpea, broad bean, rice, corn, oat, wheat, buckwheat, peanut, jordan almond, walnut, celery, Radix Dauci Sativae, potato, tomato, pears, orange, grapefruit, milk, goat milk, egg, duck's egg, goose egg, pigeon egg, pork, grass shrimp, salmon, crab in vain available from the market of farm produce, Shanghai City.The specific detection that is used for anaphylactogen lupine composition.
5 parts of lupine products (bread, cake), other does not contain 45 parts of the food of lupine composition, available from the supermarket, Shanghai, is used for actual sample and detects.
1.2 main agents and consumptive material
The LAMP reaction reagent: Bst DNA Polymerase Large Fragment, available from New England Biolabs company.
Trimethyl-glycine (Betaine), MgCl
2, available from Sigma company.
DNTPs is available from precious biotechnology (Dalian) company limited.
SYBR Green I fluorescence dye is available from Invitrogen company.
LAMP primer (forward outer primer F3, forward inner primer FIP, reverse inner primer BIP, reverse outer primer B3) entrusts Sangon Biotech (Shanghai) Co., Ltd. synthetic.
1.3 key instrument equipment
Refrigerator; Whizzer; The real-time turbidimeter of LAMP, LA-320C, Japanese Eiken Chemical; Eddy mixer; Micropipet; Analytical balance.
1.4LAMP the design of primer
Through testing widely and comparing, it is the lupine distinguished sequence that the inventor finally determines to choose the ITS1 sequence, based on this design Auele Specific Primer.External primer amplification fragment length: 236bp.
Upstream outer primer (F3, SEQ ID NO:1), downstream outer primer (B3, SEQ ID NO:2), upstream inner primer (FIP, SEQ ID NO:3), (BIP, SEQ ID NO:4) is as follows for the downstream inner primer:
F3：GAAGCCTCACAAGCAGTG；
B3：TAGGATAAGCGTGTCGCA；
1.5 sample preparation
Use refrigeration grinding machine with lupine and wheat grinds powder, and 60 ℃ of oven dry 6h.Use wheat-flour proportioning feather fan bean powder, making the lupine powder content is 1%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005% (w/w), is used for the sensitivity test of anaphylactogen lupine composition.
1.6DNA extract and preparation
Use a day root Animal genome extract a test kit (day root biochemical technology company limited, catalog number (Cat.No.): DP323) extract the DNA of animal sample, use DNA of plants extract in a small amount test kit (OMEGA company,
HP Plant DNA Kit, article No. D2485) extract the DNA of plant sample, extracting method sees the test kit process specifications for details.Use BioPhotometer plus nucleic acid-protein content meter (German Eppendorf company) to measure nucleic acid concentration.
1.7LAMP amplification and detection
LAMP reaction system: 2 * Reaction Mix (RM), 12.5 μ L, each 1.6 μ M of FIP and BIP, each 0.2 μ M of F3 and B3,20mM Tris-HCl (PH8.8), 10mM KCl, 6mM MgSO
4, 10mM (NH4)
2SO
4, 0.1%Tween20,0.8M trimethyl-glycine, 6mM MgSO
4, 1.6mM dNTP, 8U Bst large fragment DNA polysaccharase, 2 μ l DNA to be measured supply system to 25 μ l with distilled water.63 ℃ of isothermal reaction 60min, 80 ℃ of deactivation 5min finish reaction.By the real-time turbidimeter of LAMP, observe amplification curve.
Development process LAMP detects: the LAMP reaction product adds SYBR Green I fluorescence dye to observe colour-change, does not have the negative tube of amplified production to be orange, the positive pipe of amplified production is arranged for green.
1.8LAMP primer specificity experiment
To for the template of 36 kinds of animals and plants material DNA that try as the LAMP reaction, carry out the LAMP reaction, 63 ℃ of operation 60min, the specificity that checking LAMP reacts.Observe detected result with real-time turbidimeter and development process respectively.
1.9LAMP sensitivity experiment
1%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001%, 0.0005% lupine is carried out the LAMP experiment, with ddH
2O replaces DNA profiling as negative control, as positive control, carries out the LAMP amplification with lupine DNA (100ng/ μ L), and 63 ℃ of operation 60min implement turbidimeter detection reaction result, to determine to implement the LAMP detection sensitivity that turbidimeter detects.
0.1%, 0.001%, 0.0005%, 0% lupine is carried out development process LAMP experiment, with ddH
2O replaces DNA profiling as negative control, as positive control, carries out the LAMP amplification with lupine DNA (100ng/ μ L), and 63 ℃ of operation 60min are to determine development process LAMP detection sensitivity.
1.10LAMP stability experiment
With 0.1%, 0.005%, 0.001%, 0% lupine DNA is as template, each 20 parallel sample.With ddH
2O replaces DNA profiling as negative control, carries out the LAMP amplification with lupine DNA (100ng/ μ l) as positive control, 63 ℃ of operation 60min, the stability of observing the LAMP reaction.Observe detected result with real-time turbidimeter and development process respectively.
1.11 actual sample detects
To 5 parts of lupine products (bread, cake) of buying on market, other 45 portions of food that do not contain the lupine composition detect, the detect situation of checking LAMP detection method to actual sample.Detect observations with development process LAMP.
2, embodiment
The preliminary LAMP reaction system of determining 25 μ L, its composition comprises: each 1.6 μ M of FIP and BIP, each 0.2 μ M of F3 and B3,20mM Tris-HCl (PH8.8), 10mM KCl, 8mM MgSO
4, 10mM (NH
4)
2SO
4, 0.1%Tween20,1M trimethyl-glycine, 6mM MgSO
4, 1.6mM dNTP, 8U Bst large fragment DNA polysaccharase, 2 μ l DNA to be measured.63 ℃ of isothermal reaction 90min, insulation 5min finishes reaction under 80 ℃, according to appearance time and yin and yang attribute coincidence rate preliminary screening primer.Lupine standard substance with 100% carry out the preliminary screening of primer as template, simultaneously with negative DNA as negative control.The experimental result demonstration, this primer begins to occur amplification (Fig. 1) in 30min.
The optimization of LAMP reaction conditions: choose Mg
2+Concentration, trimethyl-glycine concentration, dNTPs concentration and 4 variable parameters of temperature of reaction are carried out single factors vary experiment.Mg
2+Concentration optimization: 4 Mg are set
2+Concentration gradient is followed successively by 4mM, 6mM, 8mM, 10mM; Trimethyl-glycine concentration optimization: 4 trimethyl-glycine concentration gradients are set are followed successively by 0.6M, 0.8M, 1.0M, 1.2M; DNTPs concentration optimization: 4 dNTPs concentration gradients are set are followed successively by 1.2mM, 1.4mM, 1.6mM, 1.8mM; Temperature of reaction is optimized: 4 temperature of reaction gradients are set are followed successively by 61 ℃, 63 ℃, 65 ℃, 67 ℃.When reaction is carried out, adopt the appearance time of this response curve in turbidimeter and go out peak heights as the index of estimating, above-mentioned parameter is compared.Drawing optimum parameter is: Mg
2+Concentration: 6mM; Trimethyl-glycine concentration: 0.8M; DNTPs concentration: 1.6mM; Temperature of reaction: 63 ℃.
According to above-mentioned data, set up the optimum response system, then 63 ℃ of isothermal reaction 60min finish reaction at 80 ℃ of deactivation 5min, as Fig. 2.
Embodiment 2, the experiment of food allergens lupine composition LAMP specific detection
Use the anaphylactogen primer pair to carry out the LAMP amplification for examination 36 kind of plant and animal material, detect through the real-time turbidimeter of LAMP, wherein the chrysanthemum lupine with spend lupine in vain and obvious amplification curve occurs, and amplification curve does not appear all in remaining 34 kinds of animals and plants sample, as Fig. 3.
Real-time turbidity detected result according to food allergens lupine composition LAMP specific detection, carry out development process LAMP detection to above-mentioned for the examination material, result shows: the chrysanthemum lupine is green with spending the lupine reaction solution in vain, and other animals and plants material DNA reaction solution is orange, as Fig. 4.
Turbidimeter LAMP detection in real time and development process LAMP detected result show that this LAMP primer and reaction system have species specificity, can realize specific detection.
The LAMP detection sensitivity of embodiment 3, anaphylactogen lupine composition
Pure white colored feather fan bean powder and wheat-flour are mixed into lupine content by weight is: 1%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001% and 0.0005% biased sample, be used for the LAMP detection after extracting DNA, each concentration repeats 3 times, records result with real-time turbidimeter.Turbidity LAMP detected result shows in real time, 1%, 0.1%, 0.05%, 0.01%, and amplification curve all appears in 0.005%, 0.001% lupine, and amplification curve does not appear in 0.0005% lupine.As a result, the detection of this detection method is limited to 0.001%, as Fig. 5.
According to the real-time turbidity detected result that food allergens lupine composition LAMP sensitivity detects, utilize the LAMP development process to 0.1% lupine, 0.001% lupine, 0.0005% lupine and 0% lupine sample detect.LAMP development process detected result shows, 0.1% lupine, and color reaction appears in 0.001% lupine, and color reaction does not appear in 0.0005% lupine.Therefore, the detectability of this detection method is 0.001%, as Fig. 6.
The stability experiment of the LAMP detection method of embodiment 4, anaphylactogen lupine composition
Lupine with 0.1%, 0.001% lupine product carry out real-time turbidity LAMP and detect, and each sample repeats 20 times.Turbidity LAMP test experience result shows in real time, and this detection method stable amplification all occurs for 0.1% lupine, 0.001% lupine sample, and this detection method has good stability, as Fig. 7, Fig. 9.
Lupine to 0.1%, 0.001% lupine sample carry out development process LAMP and detect, and each sample repeats 20 times.Detected result shows, color reaction all appears in minute other lupine of 20 0.1%, 0.001% lupine sample, and this detection method has good stability, as Fig. 8, Figure 10.
The application that in embodiment 5, food, lupine ultimate constituent LAMP detects
Collect 5 parts, goods (feather fan bean powder, cake, bread, Pizza etc.) containing the lupine composition on market, other does not contain 45 parts of the food (flour, bread, cake, skin Sa cake, meat intestines, jam, dumpling, beverage etc.) of lupine composition.
With newly-established detection method, these 50 samples being carried out development process LAMP detects.As a result, the detected result that 5 signs contain lupine composition sample conforms to the sample tag identifier, 45 sign do not detect the lupine composition in containing the sample of lupine composition.
Conclusion
The inventor is through further investigation, choose suitable detection gene, and based on the suitable detection reagent of this gene Selection, thereby food allergens lupine composition LAMP method for quick set up, method highly sensitive, detectability can reach 0.001% lupine; The method high specificity, with 34 kinds of sample no cross reactions of control group, operation steps is simple; detect the time limit short, accuracy of judgement as a result, the foundation of this detection method; for strengthening the food allergens identity management, improve food safety, the Protection of consumer interests are significant.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a method of identifying food allergens lupine composition, is characterized in that, described method comprises:
Take the DNA of testing sample as template, increase with the primer of specific amplification lupine ITS1 gene; If the generation specific amplification shows to comprise food allergens lupine composition in testing sample.
2. the method for claim 1, is characterized in that, described amplification is ring mediated isothermal amplification.
3. method as claimed in claim 2, it is characterized in that, the primer of described specific amplification lupine ITS1 gene comprises: upstream outer primer SEQ ID NO:1, downstream outer primer SEQ ID NO:2, upstream inner primer EQ ID NO:3, downstream inner primer SEQ ID NO:4.
4. method as claimed in claim 2, is characterized in that, described ring mediated isothermal amplification comprises: 63 ± 1 ℃ of isothermal reaction 60 ± 10min, then at 80 ± 2 ℃ of deactivation 5 ± 1min, finish reaction.
5. method as claimed in claim 2, is characterized in that, the system of described ring mediated isothermal amplification comprises primer, Mg
2+, trimethyl-glycine, dNTPs;
Wherein, Mg
2+Concentration 6 ± 1mM; Trimethyl-glycine concentration 0.8 ± 0.1M; DNTPs concentration 1.6 ± 0.2mM.
6. the method for claim 1, is characterized in that, described testing sample is food or feed.
7. a primer, is characterized in that, described primer is the LAMP amplimer, comprising: upstream outer primer SEQ ID NO:1, downstream outer primer SEQ ID NO:2, upstream inner primer EQ ID NO:3, downstream inner primer SEQ ID NO:4.
8. the purposes of primer claimed in claim 7, be used for identifying food allergens lupine composition from testing sample.
9. a test kit of identifying food allergens lupine composition, is characterized in that, comprising primer claimed in claim 7.
10. test kit as claimed in claim 9, is characterized in that, also comprises in described test kit: the examination criteria product that contain the lupine composition;
DNA extraction reagent;
The pH buffer reagent;
Potassium ion solution;
Magnesium ion solution;
Ammonium ion solution;
Tween20；
Trimethyl-glycine;
dNTP；
Bst large fragment DNA polysaccharase;
Fluorescence dye; And/or
The working instructions of the method for identifying food allergens lupine composition are described.
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| 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 |
| CN103571944A (en) * | 2013-09-23 | 2014-02-12 | 曹际娟 | Loop-mediated isothermal amplification primer of mustard component as well as kit and application thereof |
| CN114457179A (en) * | 2021-12-31 | 2022-05-10 | 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 capable of simultaneously detecting animal and plant source allergen components of food |
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Cited By (5)
| 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 |
| CN103571944A (en) * | 2013-09-23 | 2014-02-12 | 曹际娟 | Loop-mediated isothermal amplification primer of mustard component as well as kit and application thereof |
| 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 |
| CN114457179A (en) * | 2021-12-31 | 2022-05-10 | 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 capable of simultaneously detecting animal and plant source allergen components of food |
| CN114457179B (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 capable of simultaneously detecting animal and plant origin allergen components of food |
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