CN101643786A - Method for detecting allergen almond component in foods by fluorescent PCR technology - Google Patents
Method for detecting allergen almond component in foods by fluorescent PCR technology Download PDFInfo
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Abstract
The invention discloses a method for detecting an allergen almond component in foods by a fluorescent PCR technology, belonging to allergen detecting technologies, in particular a method for detectingan allergen almond component in foods by an exonuclease probe fluorescent PCR technology (TaqMan). Aiming at an allergen almond component Pru du 1.06B DNA sequence a primer and a TaqMan probe are designed, the fluorescent PCR detecting method is established. The method includes the designed primer and a probe of the almond component specificity, and the fluorescent PCR reaction condition matchedwith the primer and the probe. The method has no cross reaction with peanuts, hazelnuts, chestnuts, walnuts, pine nuts, macadamia nuts, and the like and has specificity; and the detecting sensitivitycan reach 5mg/kg. The method can be used for detecting the allergen in the foods and preventing anaphylactic reaction caused by the foods and has practical meanings.
Description
Technical field
The present invention relates to belong to the anaphylactogen detection technique, specifically use fluorescent PCR to react to detect the method for anaphylactogen almond component in the technology, particularly food of anaphylactogen.
Background technology
Food anaphylaxis is a kind of untoward reactions of people to the food generation, belongs to a kind of transformation reactions of body to exogenous material.Recent two decades comes, and food anaphylaxis is much accounted of gradually, has been considered to serious public health problem, and WHO prediction food anaphylaxis may become one of modal prevailing disease from now on.According to the U.S. FDA statistics, in the U.S., 2% grownup and 5% infant suffer from phagopyrism, and annual about 30000 people need clinical emergency treatment, and 150 people die from the anaphylaxis that food causes.In all anaphylaxis, peanut and tree nut allergy account for 10-47%.
FDA issues " food allergens sign and consumer protection bill-2004 " (FALCPA) on October 5th, 2005, stipulates that main food allergen comprises the composition of setting eight kinds of foods such as nut.Japan's label for labelling also requirement will set nut and classify as and recommend to mark composition.Use the ELISA method according to external relevant scholar and commodity are investigated, in 35 kinds of commercial goods, just have 7 kinds to contain the anaphylactogen almond component and in label, do not identify in conjunction with the PCR-ELISA method.
The domestic and international at present detection research to the anaphylactogen almond component mainly concentrates on aspects such as ELISA, PCR-ELISA, PCR, fluorescent PCR and biosensor detection.The test kit of recommending to use for the detection method FDA and the AOAC joint research of anaphylactogen has three kinds at present, is the ELISA method.But destroyed as protein in the fruit product, the ELISA method just detect less than.And DNA is than protein stabilization, and PCR method is more accurate than the ELISA method, and has avoided the demand to lot of antibodies, and European Union, Japan and AOAC advise using PCR method to prove conclusively, but do not have clear and definite specific primer sequence.
Because almond belongs to rosaceous plant together in apple, pears etc., its main allergen protein gene order has the homology of height, this research has been set up the method for anaphylactogen almond component in the utilization fluorescent PCR technology for detection food at almond component Pru du 1.06BDNA sequences Design primer and TaqMan probe.
Summary of the invention
At above-mentioned situation, the present invention has overcome shortcoming of the prior art, the method of anaphylactogen almond component in the utilization fluorescent PCR technology for detection food is provided, and is exactly that excision enzyme fluorescence probe round pcr (Taqman probe method) detects the method for anaphylactogen almond component in the food concretely.
Its technology contents comprises: the reporter plasmid that uses the employed primer of this method, probe and structure.Wherein the full sequence of primer, probe is as follows:
Primer:
The primer sequence one: TTTGGTTGAAGGAGATGCTC
The primer sequence two: TAGTTGCTGGTGCTCTTTATG
Probe:
Used probe sequence: TCCATCAGCAGATGCCACCAAC
And derive out by above-mentioned sequence, difference is not more than the oligonucleotide sequence of 8 bases and the antisense complementary sequence of each sequence, or their variant, or their partial sequence.Also comprise supporting with it fluorescent PCR reaction conditions.
The present invention is achieved by the following technical solutions:
(1) design specific oligonucleotide primer, probe with probe method probe sequence three usefulness FAM fluorophor marks, are used for excision enzyme fluorescence probe round pcr and detect;
(2) with probe method primer sequence one and probe method primer sequence two as primer, be template with the genes involved DNA of anaphylactogen almond component in the food, carry out the specific amplification of goal gene;
(3) use the fluorescent PCR instrument to carry out the real-time fluorescence luminous intensity measurement in the amplification procedure, and data transmission is crossed software kit analysis to the computer expert can observe the FAM fluorescent signal that anaphylactogen almond component in the food is carried out the specific wavelength of specific amplification generation, then prove to have anaphylactogen almond component in the food in the testing sample; Then prove and do not have anaphylactogen almond component in the food in the testing sample as not observing any fluorescent signal.
The fluorescence PCR method that this institute sets up, with nuts such as peanut, hazelnut, chestnut, walnut, pine nut, the summer, really fruits such as apple, pears, red bayberry, plum etc. did not have cross reaction, have excellent specificity, detection sensitivity can reach 5mg/kg.
Among the present invention in the fluorescent PCR reaction system each component composition as follows:
Constituent concentration application of sample amount
2 times of 25 μ L of PCR system premixture
Probe method the primer sequence one 10 μ mol/L 2 μ L
Probe method the primer sequence 2 10 μ mol/L 2 μ L
The used probe sequence one 10 μ mol/L of probe method 1.5 μ L
DNA sample 5 μ L
Distilled water 14.5 μ L
Cumulative volume 50 μ L
The fluorescent PCR amplification program is as follows
(1) 50 ℃ 2 minutes
(2) 95 ℃ 10 minutes
(3) 95 ℃ 15 seconds
(4) 60 ℃ 1 minute
(5) got back to for the 3rd step, repeat 45 times
Use the fluorescent PCR instrument to carry out the real-time fluorescence luminous intensity measurement in the amplification procedure, and data transmission is crossed software kit analysis to the computer expert can observe the FAM fluorescent signal that anaphylactogen almond component in the food is carried out the specific wavelength of specific amplification generation, then prove to have anaphylactogen almond component in the food in the testing sample; Then prove and do not have anaphylactogen almond component in the food in the testing sample as not observing any fluorescent signal.
Compared with prior art, the invention has the beneficial effects as follows: can detect owing to protein in the product is destroyed anaphylactogen almond component in the food that the ELISA method can not detect.The present invention's method amplified target gene of PCR, the complex process of having avoided ELISA to react saves time; Other proteic interference that the PCR authentication method is subjected to are less, and are more accurate than the ELISA method.
Description of drawings
Fig. 1 almond is added into chocolate samples DNA, fluorescence signal intensity.
Fig. 2 buys international almond anaphylactogen reference material DNA, fluorescence signal intensity.
Other nuts of the non-almond of Fig. 3 and Rosaceae other plant DNA, fluorescence signal intensity.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.
Sample: the chocolate that does not contain almond component is cooked interpolation matrix.Get almond that 1g grinds to form pasty state and add in the 99g chocolate 40 ℃ of water-baths and melt, stir 30min, thorough mixing is even.The chocolate of getting the above-mentioned interpolation almond of 3g adds in the 27g chocolate, with this continuous dilution, finally makes the chocolate that contains almond 1000mg/kg, 100mg/kg, 20mg/kg, 10mg/kg, 5mg/kg.
1. sample preparation
(1) gets the 300mg sample respectively, put into the 1.5mL centrifuge tube, add 600 μ L CTAB damping fluids (CTAB 55mmol/L, EDTA20mmol/L, Tris 100mmol/L, 10% hydrochloric acid adjust pH to 8.0), 15 μ L (20mg/ml) Proteinase Ks, 65 ℃ of incubation 30min; Add 500 μ L phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixed solution vibrates strongly, centrifugal 12000rpm 15min; Draw supernatant liquor and add the equal-volume Virahol, the strong vibration centrifugal 12000rpm in back, 10min abandons supernatant; With 200 μ LTE dissolving (the TE amount decides what on the DNA precipitation); Add the equal-volume chloroform: primary isoamyl alcohol (24: 1) mixed solution vibrates strongly, centrifugal 12000rpm 15min; Draw supernatant liquor and add the equal-volume Virahol, the strong vibration centrifugal 12000rpm in back, 10min abandons supernatant; Dissolve with 200 μ LTE.Dissolved solution repeats the above-mentioned steps purify DNA as sample.Detect its purity and concentration with the DNA analysis instrument.
2.PCR amplification
Constituent concentration application of sample amount
2 times of 25 μ L of PCR system premixture
Probe method the primer sequence one 10 μ mol/L 2 μ L
Probe method the primer sequence 2 10 μ mol/L 2 μ L
The used probe sequence one 10 μ mol/L of probe method 1.5 μ L
DNA sample 5 μ L
Distilled water 14.5 μ L
Cumulative volume 50 μ L
The fluorescent PCR amplification program is as follows
(1) 50 ℃ 2 minutes
(2) 95 ℃ 10 minutes
(3) 95 ℃ 15 seconds
(4) 60 ℃ 1 minute
(5) got back to for the 3rd step, repeat 45 times
PCR carries out 7 pipe PCR experiments altogether, and wherein the DNA sample is added is respectively that to contain the filbert composition be 1000mg/kg, 100mg/kg, 20mg/kg, 10mg/kg, 5mg/kg sample, blank and positive control.
3. detected fluorescent PCR collection of illustrative plates is observed
Each concentration sample be can observe in the fluorescent PCR process and tangible FAM fluorescence, result such as Fig. 1 produced in 23.2,35.2,37.0,38.0,39.0,39.1 circulations respectively.
The FAM fluorescence intensity signals of each the bar curve representative among Fig. 1 is respectively:
1. positive control;
2. contain the chocolate DNA sample of almond component 1000mg/kg;
3. contain the chocolate DNA sample of almond component 100mg/kg;
4. contain the chocolate DNA sample of almond component 20mg/kg;
5. contain the chocolate DNA sample of almond component 10mg/kg;
6. contain the chocolate DNA sample of almond component 5mg/kg;
7. blank;
Experiment shows that this method detection sensitivity reaches 5mg/kg.
Embodiment 2
Sample: international almond anaphylactogen reference material 40mg/kg, 20mg/kg, 10mg/kg, 5mg/kg
1. sample preparation
(1) gets the 300mg sample respectively, put into the 1.5mL centrifuge tube, add 600 μ L CTAB damping fluid (CTAB 55mmol/L, EDTA 20mmol/L, Tris 100mmol/L, 10% hydrochloric acid adjust pH to 8.0), 15 μ L (20mg/ml) Proteinase Ks,, 65 ℃ of incubation 30min; Add 500 μ L phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixed solution vibrates centrifugal 12000rpm15min strongly; Draw supernatant liquor and add the equal-volume Virahol, the strong vibration centrifugal 12000rpm in back, 10min abandons supernatant; With 200 μ LTE dissolving (the TE amount decides what on the DNA precipitation); Add the equal-volume chloroform: primary isoamyl alcohol (24: 1) mixed solution vibrates strongly, centrifugal 12000rpm 15min; Draw supernatant liquor and add the equal-volume Virahol, the strong vibration centrifugal 12000rpm in back, 10min abandons supernatant; Dissolve with 200 μ L TE.Dissolved solution repeats the above-mentioned steps purify DNA as sample.Detect its purity and concentration with the DNA analysis instrument.
2.PCR amplification
Constituent concentration application of sample amount
2 times of 25 μ L of PCR system premixture
Probe method the primer sequence one 10 μ mol/L 2 μ L
Probe method the primer sequence 2 10 μ mol/L 2 μ L
The used probe sequence one 10 μ mol/L of probe method 1.5 μ L
DNA sample 5 μ L
Distilled water 14.5 μ L
Cumulative volume 50 μ L
The fluorescent PCR amplification program is as follows
(1) 50 ℃ 2 minutes
(2) 95 ℃ 10 minutes
(3) 95 ℃ 15 seconds
(4) 60 ℃ 1 minute
(5) got back to for the 3rd step, repeat 45 times
PCR carries out 5 pipe PCR experiments altogether, wherein the DNA and the blank of the difference 40mg/kg, the 20mg/kg that are added of DNA sample, 10mg/kg, 5mg/kg reference material.
3. detected fluorescent PCR collection of illustrative plates is observed
Each concentration sample be can observe in the fluorescent PCR process and tangible FAM fluorescence, result such as Fig. 2 produced in 32.2,34.6,35.1,36.8 circulations respectively.
The FAM fluorescence intensity signals of each the bar curve representative among Fig. 2 is respectively:
1. the reference material DNA sample that contains the 40mg/kg almond component;
2. the reference material DNA sample that contains the 20mg/kg almond component;
3. the reference material DNA sample that contains the 10mg/kg almond component;
4. the reference material DNA sample that contains the 5mg/kg almond component;
5. blank.
Experiment shows that this method detection sensitivity reaches the conclusive evidence that 5mg/kg obtains international reference material.
Embodiment 3
Sample: almond; Nuts such as peanut, hazelnut, chestnut, walnut, pine nut, summer are really; Fruits such as apple, pears, red bayberry, plum.
1. sample preparation
(1) gets the 300mg sample respectively, put into the 1.5mL centrifuge tube, add 600 μ L CTAB damping fluid (CTAB 55mmol/L, EDTA 20mmol/L, Tris 100mmol/L, 10% hydrochloric acid adjust pH to 8.0), 15 μ L (20mg/ml) Proteinase Ks,, 65 ℃ of incubation 30min; Add 500 μ L phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixed solution vibrates centrifugal 12000rpm15min strongly; Draw supernatant liquor and add the equal-volume Virahol, the strong vibration centrifugal 12000rpm in back, 10min abandons supernatant; With 200 μ L TE dissolving (the TE amount decides what on the DNA precipitation); Add the equal-volume chloroform: primary isoamyl alcohol (24: 1) mixed solution vibrates strongly, centrifugal 12000rpm 15min; Draw supernatant liquor and add the equal-volume Virahol, the strong vibration centrifugal 12000rpm in back, 10min abandons supernatant; Dissolve with 200 μ L TE.Dissolved solution repeats the above-mentioned steps purify DNA as sample.Detect its purity and concentration with the DNA analysis instrument.
2.PCR amplification
Constituent concentration application of sample amount
2 times of 25 μ L of PCR system premixture
Probe method the primer sequence one 10 μ mol/L 2 μ L
Probe method the primer sequence 2 10 μ mol/L 2 μ L
The used probe sequence one 10 μ mol/L of probe method 1.5 μ L
DNA sample 5 μ L
Distilled water 14.5 μ L
Cumulative volume 50 μ L
The fluorescent PCR amplification program is as follows
(1) 50 ℃ 2 minutes
(2) 95 ℃ 10 minutes
(3) 95 ℃ 15 seconds
(4) 60 ℃ 1 minute
(5) got back to for the 3rd step, repeat 45 times
PCR carries out 11 pipe PCR experiments, the original content DNA sample that is respectively testing sample that wherein the DNA sample is added altogether.
3. detected fluorescent PCR collection of illustrative plates is observed
Almond DNA be can observe in the fluorescent PCR process and tangible FAM fluorescence, result such as Fig. 3 produced in 30.2 circulations.The FAM fluorescence intensity signals of the curve representative among Fig. 3 is respectively the original content DNA sample of testing sample.
Experiment shows this method high specificity.
Sequence list
SEQUENCE?LISTING
＜110〉Animal-Plant and food Detecting Center, Tianjin Exit-Entery Inspection ﹠ Quarant
＜120〉a kind of method of using anaphylactogen almond component in the fluorescent PCR technology for detection food
<140>200910068848.7
<141>2009-05-15
<160>3
<170>PatentIn?version?3.5
<210>1
<211>20
<212>DNA
＜213〉synthetic
<220>
<221>prim_bind
<222>(1)..(20)
<400>1
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<212>DNA
＜213〉synthetic
<220>
<221>prim_bind
<222>(1)..(21)
<400>2
<210>3
<211>22
<212>DNA
＜213〉synthetic
<220>
<221>prim_bind
<222>(1)..(22)
<400>3
Claims (5)
1. a method of using anaphylactogen almond component in the fluorescent PCR technology for detection food is characterized in that, a kind of fluorescent PCR technology is meant that excision enzyme fluorescence probe round pcr (TaqMan) probe technique detects the method for anaphylactogen almond component in the food.
2. a kind of method of using anaphylactogen almond component in the fluorescent PCR technology for detection food according to claim 1, it is characterized in that the sequence of the employed primer of method, probe that excision enzyme fluorescence probe round pcr (TaqMan probe method) detects almond is as follows:
Primer:
The primer sequence one: TTTGGTTGAAGGAGATGCTC
The primer sequence two: TAGTTGCTGGTGCTCTTTATG
Probe:
Used probe sequence: TCCATCAGCAGATGCCACCAAC
3. according to the designed primer sequence of claim 2, probe sequence, it is characterized in that, also comprise derive out by above-mentioned three primers and probe, difference is not more than the oligonucleotide sequence of 8 bases and the antisense complementary sequence of each sequence, or their variant, or their partial sequence.
4. a kind of method of using anaphylactogen almond component in the fluorescent PCR technology for detection food according to claim 1 is characterized in that each component composition is as follows in the PCR reaction system:
Constituent concentration application of sample amount
2 times of 25 μ L of PCR system premixture
Probe method the primer sequence one 10 μ mol/L 2 μ L
Probe method the primer sequence 2 10 μ mol/L 2 μ L
The used probe sequence one 10 μ mol/L of probe method 1.5 μ L
DNA sample 5 μ L
Distilled water 14.5 μ L
Cumulative volume 50 μ L
5. a kind of method of using anaphylactogen almond component in the fluorescent PCR technology for detection food according to claim 1 is characterized in that amplification program is in the PCR method
(1) 50 ℃ 2 minutes
(2) 95 ℃ 10 minutes
(3) 95 ℃ 15 seconds
(4) 60 ℃ 1 minute
(5) got back to for the 3rd step, repeat 45 times.
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| CN102344951A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer, method and kit for detecting pear-derived components in sample |
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| CN103031377A (en) * | 2012-12-14 | 2013-04-10 | 郑秋月 | Real-time fluorescence PCR (Polymerase Chain Reaction) detection method for components of apricot in food and beverage |
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| CN104569171A (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 | Almond mass spectrometric detection characteristic sequence group and detection kit |
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| CN101109750A (en) * | 2007-07-27 | 2008-01-23 | 杭州浙大生科生物技术有限公司 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
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| CN102344951A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer, method and kit for detecting pear-derived components in sample |
| CN102344953A (en) * | 2010-07-30 | 2012-02-08 | 中国检验检疫科学研究院 | Primer for detecting peach-derived component in sample, method and kit |
| CN102344952B (en) * | 2010-07-30 | 2013-06-19 | 中国检验检疫科学研究院 | Primer, method and kit for detecting apple-derived materials in sample |
| CN102344953B (en) * | 2010-07-30 | 2013-07-24 | 中国检验检疫科学研究院 | Primer for detecting peach-derived component in sample, method and kit |
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| CN103031377A (en) * | 2012-12-14 | 2013-04-10 | 郑秋月 | Real-time fluorescence PCR (Polymerase Chain Reaction) detection method for components of apricot in food and beverage |
| CN103031377B (en) * | 2012-12-14 | 2014-05-07 | 郑秋月 | Real-time fluorescence PCR (Polymerase Chain Reaction) detection method for components of apricot in food and beverage |
| CN103060461A (en) * | 2013-01-18 | 2013-04-24 | 天津生物芯片技术有限责任公司 | Specific primer and kit for detecting common food allergens |
| CN104569171A (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 | Almond mass spectrometric detection characteristic sequence group and detection kit |
| CN109295250A (en) * | 2018-10-31 | 2019-02-01 | 四川华汉三创生物科技有限公司 | A kind of detection kit and method of food-borne plant hypersensitive ultimate constituent |
| CN109295250B (en) * | 2018-10-31 | 2022-03-22 | 四川华汉三创生物科技有限公司 | Detection kit and method for food-borne plant allergen components |
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