CN104419761A - Detection kit for buckwheat allergen ingredients by employing loop-mediated isothermal amplification and application method of kit - Google Patents
Detection kit for buckwheat allergen ingredients by employing loop-mediated isothermal amplification and application method of kit Download PDFInfo
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- CN104419761A CN104419761A CN201310407049.4A CN201310407049A CN104419761A CN 104419761 A CN104419761 A CN 104419761A CN 201310407049 A CN201310407049 A CN 201310407049A CN 104419761 A CN104419761 A CN 104419761A
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Abstract
The invention belongs to a technology for detecting buckwheat allergen ingredients in food, and particularly relates to a detection kit for buckwheat allergen in food by employing a multi-loop-mediated isothermal amplification technology and an application method of the detection kit. The kit and the detection method have the advantages of rapidness, high specificity, high sensitivity, good repeatability and the like, is simple to operate and can be widely applied to the field of rapid detection of the buckwheat allergen ingredients in food.
Description
Technical field
The present invention relates to one utilizes many ring mediated isothermal amplifications (LAMP) technology to carry out the method for quick of buckwheat allergen composition in food, specifically the preparation and application of buckwheat allergen composition quick detection kit in a kind of food.
Background technology
Buckwheat is also called triangle wheat, Wu Mai, Hua Qiao, belong to dicotyledonous polygonaceae plant, its Cultivar is sweet cherry roots (Fagopyrum esculentum, Common buckwheat, be called for short CB) and Radix Et Rhizoma Fagopyri Tatarici (Fagopyrum tataricum, Tartary buckwheat, is called for short TB).Buckwheat has higher nutritive value and pharmaceutical use, containing multiple human body essential amino acid and biologically active substance, can prevent and treat the various diseases such as diabetes, hypertension, coronary heart disease and stomach trouble, have fabulous health-care effect to human body.But the anaphylactogen that buckwheat contains becomes branch to make many contacts or its people edible produce allergic symptom, and the buckwheat components even containing trace in food all can cause the anaphylaxis such as serious shock.
Therefore, urgent need sets up buckwheat allergic component method for quick and related detecting method standard in food, this research adopts ring mediated isothermal amplification (lamp) method to detect buckwheat allergen composition in food, set up the method for buckwheat allergic component (cultivation type and wild-type) in efficient, special, rapid detection food, the qualification for import and export food buckwheat components provides practicable technical support.
Summary of the invention
The object of this invention is to provide a kind of fast, high specificity, the technological method that highly sensitive and cost is low, mainly use loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) method detects the test kit of buckwheat allergen composition in food, and provide the preparation and application of buckwheat allergen composition in a kind of food, thus provide scientific basis for the rapid detection of buckwheat allergen composition in food.
Cardinal principle of the present invention is: ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP), it is a kind of novel nucleic acid amplification method, be characterized in 6 zone design 4 species-specific primers for target gene, under the effect of strand displacement archaeal dna polymerase (Bst DNA polymerasc), 60-65 DEG C of constant-temperature amplification, about 15-60rain gets final product nucleic acid amplification, and efficiency can reach 10
9 ~ 10the individual order of magnitude, has the features such as simple to operate, high specificity, product easily detect.
A kind of buckwheat allergen composition of the present invention loop-mediated isothermal amplification detection kit, is characterized in that comprising:
(1) LAMP amplification reaction solution A
Comprise: 10 × ThermolPol damping fluid, 50mmol/L Adlerika, 5mol/L trimethyl-glycine, dNTPs solution (often kind of nucleotide concentration 10 mmol/L), 8U/ μ L
bstarchaeal dna polymerase, upstream and downstream primer outside 0.2-0.6 μm of ol/L, upstream and downstream primer inside 0.2-0.6 μm of ol/L;
Described 10 × ThermolPol damping fluid contains 200 mmol/L Tris-HCl(pH 8.8), 100 mmol/L ammonium sulfate, 100 mmol/L Repone K, 20 mmol/L magnesium sulfate, 1% Triton X-100;
Described outside upstream primer (F3) sequence is: 5 '-ATCCTCGGACCGAGATGG-3 ', outside downstream primer (B3) sequence is: 5 '-AGTCCTTCTCTTCCTGCCT-3 ', inner side upstream primer (FIP) sequence is: 5 '-CTCCGACAACCTGGACTCTTCCAACTTGAACGCGCACAGC-3 ', and inner side downstream primer (BIP) sequence is 5 '-AGTGTGTTCGACGACAACGTGCTCAACACCACTGCGAATCC-3 ';
The mass ratio of described dNTP tetra-kinds of thymus nucleic acids is dUTP:dATP:dGTP:dCTP=2:1:1:1.
(2) nitrite ion: SYBR Green I fluorescence dye or DNAGreen, 1000 ×;
LAMP amplification reaction solution A often pipe 23ul, consist of: 2.5ul 10 × ThermoPol damping fluid, upstream primer (F3) outside 1.0ul 5 μm of ol/L, downstream primer (B3) outside 1.0ul 5 μm of ol/L, upstream primer (FIP) inside 1.0 ul 40 μm ol/L, downstream primer (BIP) inside 1.0ul 40 μm of ol/L, 5.0ul 10 mmol/L dNTPs, 4ul 5 mol/L trimethyl-glycine, 0.5ul 100 mmol/L Adlerika (comprising institute's containing magnesium sulfate in damping fluid).1ul 8 U/ μ L Bst archaeal dna polymerase.2ul 10 ng/ μ L DNA profiling and 6ul distilled water.
A using method for buckwheat allergen composition loop-mediated isothermal amplification detection kit, is characterized in that comprising the following steps:
(1) extraction of measuring samples DNA:
A. take the pretreated sample of 300 mg in 2 mL centrifuge tubes, add 600 μ L CTAB damping fluids and 40 μ L Proteinase Ks, vibration mixing, 65 DEG C of temperature bath 30 min ~ 90 min, period mixes every 10 min gentle inversion;
B. centrifugal 5 min of 2 000g, get supernatant in another 2 clean mL centrifuge tubes, add the mixed solution (25:24:1) of isopyknic phenol, trichloromethane and primary isoamyl alcohol, concussion mixing;
C. centrifugal 10 min of 12 000 g, get supernatant in 2 clean mL centrifuge tubes, add equal-volume Virahol, put upside down mixing;
D. centrifugal 10 min of 12 000 g, abandoning supernatant, with the TE buffer solution DNA being preheated to 65 DEG C;
E. 5 μ L RNA enzyme solution are added, 37 DEG C of 30 min;
F. 200 μ L trichloromethanes are added: primary isoamyl alcohol (24:1), intense oscillations;
G. centrifugal 10 min of 12 000 g, get supernatant in 2 clean mL centrifuge tubes, add equal-volume Virahol, put upside down mixing;
H. centrifugal 10 min of 12 000 g, abandoning supernatant, with 70% ethanol 500 μ L of 4 DEG C of precoolings, vortex washing and precipitating;
I. centrifugal 10 min of 12 000 g, abandoning supernatant, be inverted after drying and add 100 μ L TE buffer solution precipitations, be measuring samples template DNA:
(2) loop-mediated isothermal amplification of buckwheat allergen composition is carried out
A: add 2ul measuring samples template DNA in the reaction tubes that 23uL LAMP reaction solution A is housed.
B:65 DEG C of constant-temperature amplification 60 min, 80 DEG C of 5 min makes enzyme-deactivating, and reaction terminates.
(3) color developing detection
After reaction terminates, 1ul nitrite ion is added in each reaction tubes to be checked, nitrite ion and reaction solution are turned upside down and mixes gently, color observation is carried out immediately under black background, if color is yellow, measuring samples is described not containing buckwheat allergen composition, if color becomes green, then illustrates that measuring samples contains buckwheat allergen composition.
The invention has the advantages that and establish buckwheat allergen composition loop-mediated isothermal amplification detection kit and detection method in food, this test kit is two specific inner primers and two specificity outer primers according to conserved regions six sequences Design of buckwheat allergen albumen, this conserved genetic sequences is that the main kind of buckwheat is common, to ensure that the level from planting detects buckwheat allergen composition.The present invention adopts loop-mediated isothermal amplification technique, this technology high specificity, identical highly sensitive is had with PCR detection method, and result need not be observed with gel electrophoresis, fluorescence dye is used to observe, simple and quick, can be used for the detection of buckwheat allergen composition in food, be applicable to basic unit and detect use.
embodiment:
The detection kit of the buckwheat allergen composition made by following formula includes following (1)-(2):
(1) LAMP amplification reaction solution A
Comprise 10 × ThermolPol damping fluid, 50mmol/L Adlerika, 5mol/L trimethyl-glycine, dNTPs solution (often kind of nucleotide concentration 10 mmol/L), 8U/ μ L
bstarchaeal dna polymerase, upstream and downstream primer outside 0.3 μm of ol/L, upstream and downstream primer inside 0.3 μm of ol/L.
Wherein 10 × ThermolPol damping fluid contains 200 mmol/L Tris-HCl(pH 8.8), 100 mmol/L ammonium sulfate, 100 mmol/L Repone K, 20 mmol/L magnesium sulfate, 1% Triton X-100;
Wherein said outside upstream primer (F3) sequence is: 5 '-ATCCTCGGACCGAGATGG-3 ', outside downstream primer (B3) sequence is: 5 '-AGTCCTTCTCTTCCTGCCT-3 ', inner side upstream primer (FIP) sequence is: 5 '-CTCCGACAACCTGGACTCTTCCAACTTGAACGCGCACAGC-3 ', and inner side downstream primer (BIP) sequence is 5 '-AGTGTGTTCGACGACAACGTGCTCAACACCACTGCGAATCC-3 ';
(2) nitrite ion: SYBR Green I fluorescence dye or DNAGreen, 1000 ×.
Above-mentioned ring isothermal amplification liquid A often the consisting of of pipe 23ul: 2.5ul 10 × ThermoPol damping fluid, upstream primer (F3) outside 1.0ul 5 μm of ol/L, downstream primer (B3) outside 1.0ul 5 μm of ol/L, upstream primer (FIP) inside 1.0 ul 40 μm ol/L, downstream primer (BIP) inside 1.0ul 40 μm of ol/L, 5.0ul 10 mmol/L dNTPs, 4ul 5 mol/L trimethyl-glycine, 0.5ul 100 mmol/L Adlerika (comprising institute's containing magnesium sulfate in damping fluid).1ul 8 U/ μ L Bst archaeal dna polymerase.2ul 10 ng/ μ L DNA profiling and 6ul distilled water.
Use mentioned reagent box to detect buckwheat allergen composition to detect according to following program:
(1) extraction of measuring samples DNA:
A. take the pretreated sample of 300 mg in 2 mL centrifuge tubes, add 600 μ L CTAB damping fluids and 40 μ L Proteinase Ks, vibration mixing, 65 DEG C of temperature bath 60min, period mixes every 10 min gentle inversion;
B. centrifugal 5 min of 2 000g, get supernatant in another 2 clean mL centrifuge tubes, add the mixed solution (25:24:1) of isopyknic phenol, trichloromethane and primary isoamyl alcohol, concussion mixing;
C. centrifugal 10 min of 12 000 g, get supernatant in 2 clean mL centrifuge tubes, add equal-volume Virahol, put upside down mixing;
D. centrifugal 10 min of 12 000 g, abandoning supernatant, with the TE buffer solution DNA being preheated to 65 DEG C;
E. 5 μ L RNA enzyme solution are added, 37 DEG C of 30 min;
F. 200 μ L trichloromethanes are added: primary isoamyl alcohol (24:1), intense oscillations
G. centrifugal 10 min of 12 000 g, get supernatant in 2 clean mL centrifuge tubes, add equal-volume Virahol, put upside down mixing
H. centrifugal 10 min of 12 000 g, abandoning supernatant, with 70% ethanol 500 μ L of 4 DEG C of precoolings, vortex washing and precipitating;
I. centrifugal 10 min of 12 000 g, abandoning supernatant, be inverted after drying and add 100 μ L TE buffer solution precipitations, be measuring samples template DNA:
(2) loop-mediated isothermal amplification of buckwheat allergen composition is carried out:
A: add 2ul measuring samples template DNA in the reaction tubes that 23uL LAMP reaction solution A is housed.
B:65 DEG C of constant-temperature amplification 60 min, 80 DEG C of 5 min makes enzyme-deactivating, and reaction terminates.
(3) color developing detection:
After reaction terminates, 1ul nitrite ion is added in each reaction tubes to be checked, nitrite ion and reaction solution are turned upside down and mixes gently, color observation is carried out immediately under black background, if color is yellow, measuring samples is described not containing buckwheat allergen composition, if color becomes green, then illustrates that measuring samples contains buckwheat allergen composition.
Claims (5)
1. a buckwheat allergen composition loop-mediated isothermal amplification detection kit, is characterized in that comprising:
(1) LAMP amplification reaction solution A
Comprise: 10 × ThermolPol damping fluid, 50mmol/L Adlerika, 5mol/L trimethyl-glycine, dNTPs solution (often kind of nucleotide concentration 10 mmol/L), 8U/ μ L
bstarchaeal dna polymerase, upstream and downstream primer outside 0.2-0.6 μm of ol/L, upstream and downstream primer inside 0.2-0.6 μm of ol/L;
Described outside upstream primer (F3) sequence is: 5 '-ATCCTCGGACCGAGATGG-3 ', outside downstream primer (B3) sequence is: 5 '-AGTCCTTCTCTTCCTGCCT-3 ', inner side upstream primer (FIP) sequence is: 5 '-CTCCGACAACCTGGACTCTTCCAACTTGAACGCGCACAGC-3 ', and inner side downstream primer (BIP) sequence is 5 '-AGTGTGTTCGACGACAACGTGCTCAACACCACTGCGAATCC-3 ';
(2) nitrite ion: SYBR Green I fluorescence dye or DNAGreen, 1000 ×.
2., according to the buckwheat allergen composition loop-mediated isothermal amplification detection kit described in claim 1, it is characterized in that described 10 × ThermolPol damping fluid contains 200 mmol/L Tris-HCl(pH 8.8), 100 mmol/L ammonium sulfate, 100 mmol/L Repone K, 20 mmol/L magnesium sulfate, 1% Triton X-100.
3., according to the buckwheat allergen composition loop-mediated isothermal amplification detection kit described in claim 1, it is characterized in that the mass ratio of described dNTP tetra-kinds of thymus nucleic acids is dUTP:dATP:dGTP:dCTP=2:1:1:1.
4. according to a kind of buckwheat allergen composition loop-mediated isothermal amplification detection kit described in claim 1, it is characterized in that described LAMP amplification reaction solution A often pipe 23ul, consist of: 2.5ul 10 × ThermoPol damping fluid, upstream primer (F3) outside 1.0ul 5 μm of ol/L, downstream primer (B3) outside 1.0ul 5 μm of ol/L, upstream primer (FIP) inside 1.0 ul 40 μm ol/L, downstream primer (BIP) inside 1.0ul 40 μm of ol/L, 5.0ul 10 mmol/L dNTPs, 4ul 5 mol/L trimethyl-glycine, 0.5ul 100 mmol/L Adlerika (comprising institute's containing magnesium sulfate in damping fluid), 1ul 8 U/ μ L Bst archaeal dna polymerase, 2ul 10 ng/ μ L DNA profiling and 6ul distilled water.
5. a using method for buckwheat allergen composition loop-mediated isothermal amplification detection kit, is characterized in that comprising the following steps:
(1) extraction of measuring samples DNA
A. take the pretreated sample of 300 mg in 2 mL centrifuge tubes, add 600 μ L CTAB damping fluids and 40 μ L Proteinase Ks, vibration mixing, 65 DEG C of temperature bath 30 min ~ 90 min, period mixes every 10 min gentle inversion;
B. centrifugal 5 min of 2 000g, get supernatant in another 2 clean mL centrifuge tubes, add the mixed solution (25:24:1) of isopyknic phenol, trichloromethane and primary isoamyl alcohol, concussion mixing;
C. centrifugal 10 min of 12 000 g, get supernatant in 2 clean mL centrifuge tubes, add equal-volume Virahol, put upside down mixing;
D. centrifugal 10 min of 12 000 g, abandoning supernatant, with the TE buffer solution DNA being preheated to 65 DEG C;
E. 5 μ L RNA enzyme solution are added, 37 DEG C of 30 min;
F. 200 μ L trichloromethanes are added: primary isoamyl alcohol (24:1), intense oscillations;
G. centrifugal 10 min of 12 000 g, get supernatant in 2 clean mL centrifuge tubes, add equal-volume Virahol, put upside down mixing;
H. centrifugal 10 min of 12 000 g, abandoning supernatant, with 70% ethanol 500 μ L of 4 DEG C of precoolings, vortex washing and precipitating;
I. centrifugal 10 min of 12 000 g, abandoning supernatant, is inverted after drying and adds 100 μ L TE buffer solution precipitations, be measuring samples template DNA;
(2) loop-mediated isothermal amplification of buckwheat allergen composition is carried out
A: add 2ul measuring samples template DNA in the reaction tubes that 23uL LAMP reaction solution A is housed;
B:65 DEG C of constant-temperature amplification 60 min, 80 DEG C of 5 min makes enzyme-deactivating, and reaction terminates;
(3) color developing detection
After reaction terminates, 1ul nitrite ion is added in each reaction tubes to be checked, nitrite ion and reaction solution are turned upside down and mixes gently, color observation is carried out immediately under black background, if color is yellow, measuring samples is described not containing buckwheat allergen composition, if color becomes green, then illustrates that measuring samples contains buckwheat allergen composition.
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| CN201310407049.4A CN104419761A (en) | 2013-09-10 | 2013-09-10 | Detection kit for buckwheat allergen ingredients by employing loop-mediated isothermal amplification and application method of kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755298A (en) * | 2016-11-15 | 2017-05-31 | 河南省产品质量监督检验院 | It is a kind of that quantitative detection method is carried out to original soybean sensitive P34 GFPs in food |
| CN106916896A (en) * | 2016-04-27 | 2017-07-04 | 苏茂顺 | A kind of kit and detection method for detecting ergot |
| CN107299142A (en) * | 2017-07-26 | 2017-10-27 | 上海速创诊断产品有限公司 | A kind of LAMP primer composition thing and its kit for being used to detect sesame anaphylactogen 2S albumin genes |
-
2013
- 2013-09-10 CN CN201310407049.4A patent/CN104419761A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106916896A (en) * | 2016-04-27 | 2017-07-04 | 苏茂顺 | A kind of kit and detection method for detecting ergot |
| CN106916896B (en) * | 2016-04-27 | 2018-04-13 | 广州博疆一生物科技有限公司 | A kind of kit and detection method for being used to detect ergot |
| CN106755298A (en) * | 2016-11-15 | 2017-05-31 | 河南省产品质量监督检验院 | It is a kind of that quantitative detection method is carried out to original soybean sensitive P34 GFPs in food |
| CN107299142A (en) * | 2017-07-26 | 2017-10-27 | 上海速创诊断产品有限公司 | A kind of LAMP primer composition thing and its kit for being used to detect sesame anaphylactogen 2S albumin genes |
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Application publication date: 20150318 |