CN103540673A - Dual PCR (Polymerase Chain Reaction) detection method for soybean and peanut components in walnut milk beverage - Google Patents
Dual PCR (Polymerase Chain Reaction) detection method for soybean and peanut components in walnut milk beverage Download PDFInfo
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Abstract
The invention discloses a method for simultaneously detecting soybean and peanut components doped in walnut milk by using a dual PCR (Polymerase Chain Reaction) technology. According to the method, a specific primer is designed aiming at the soybean and peanut components so as to establish a dual PCR detection method for main doped components, namely the soybean and peanut components, in the walnut milk. The dual PCR detection method comprises the specific primer aiming at the soybean and peanut components and a PCR reaction condition matched with the specific primer; the primer designed by the method has no crossed reactions with other common leguminous plants and non-leguminous plants including peanuts, filberts, walnuts, pine nuts, pistachio nuts, carya illinoensis koch, cashew nuts, almonds, sesames, apples and the like, and has the good specificity; the method disclosed by the invention can be used for simultaneously detecting the soybean and peanut components which are mainly doped in the walnut milk; the result is obtained rapidly, accurate and reliable; the method has an actual application value for detecting that soybean and peanut proteins in the market are used for replacing walnut proteins. Meanwhile, the method has an important reference value for rapid and specific detection when other low-price plant proteins are used for replacing the walnut proteins and relative milk products.
Description
Technical field
The invention belongs to Safety of Food Quality monitoring technical field, method for quick when relating in particular to application double PCR method to the soybean of mixing in Walnut Milk drink, peanut composition.
Background technology
Along with the raising day by day of people's living standard, increasing to the demand of health-care nutritional food; Along with the transformation of people's consumption consciousness, the consumption consciousness of pursuing " nutrition, safety, health " is also denseer.Walnut is a kind of nutritious food raw material, not containing cholesterol, contain abundant riboflavin, Yelkin TTS, trace element, VITAMIN, amino acid and a large amount of unsaturated fatty acids, can prevent body early ageing, promote brain cell development, reduce cholesterol synthesize, prevent arteriosclerosis, be a kind of desirable nutritive health-care food.And in walnut protein, contain the multiple amino acids composition useful to human body, and walnut beverage relies on the advantage of its health, nutrition, safety, fashion to obtain fast development, and the industrialized development of Walnut Milk drink attracts people's attention, and market outlook are very wide.Take food that walnut is main component undressed before, according to form, be easy to walnut and other species to differentiate, but after food-processing is processed, often losing intuitively can diagnostic characteristics.Illegal businessman is for pursuing immediate interest, adopt soybean, the peanut of relative low price to copy walnut beverage, adulterate and fight for market with low price, serious infringement human consumer's legitimate rights and interests, that even gives human consumer healthy and safely brings threat, has also had a strong impact on the sound development of Walnut Industry.The examination criteria that Walnut Milk (dew) is carried out at present, with sense organ and the various physics and chemistry value of physics and chemistry method detection Walnut Milk, as: protein, fat etc., though can make preliminary judgement to it, theoretical foundation is insufficient, can not adapt to the needs of social development now.In bibliographical information and national corresponding plants protein drinks standard, all do not have simultaneously mixing soybean in Walnut Milk, Semen arachidis hypogaeae protein carries out the effective ways of differentiating fast.
At present domestic the evaluation containing walnut protein ingredient in walnut composition food is mainly and with antibody-antigen-specific, is identified as basic Enzyme-linked Immunosorbent Assay (ELISA) and detects, for mixing common low price albumen, have not yet to see report as the DNA detection of soybean, Semen arachidis hypogaeae protein.Due in the production process of Walnut Milk, in drink, have a small amount of residual starting material DNA, and DNA is than protein stabilized, so PCR method is more accurate compared with ELISA method, and has avoided the demand to lot of antibodies.This research, for soybean, peanut Composition Design Auele Specific Primer, has been set up and has been used double PCR technology to detect the method for mixing soybean, peanut composition in Walnut Milk simultaneously.
Summary of the invention
For above-mentioned situation, the present invention has overcome shortcoming of the prior art, object is just to set up a kind of method of using round pcr simultaneously to detect fast main dopant soybean and peanut composition in Walnut Milk, for the discriminating of adulterated composition in all kinds of walnut protein food and healthcare products provides technical support.For achieving the above object, method of the present invention is: for soybean, design a group-specific primers Bd 28K-F and Bd 28K-R; For peanut, design group-specific primers Arah1-F and an Arah1-R; Extract the DNA of different plant dairy products, utilize two pairs of primers to carry out double PCR amplification, primer is carried out to the checking of species specificity, observe and whether have soybean specific amplification band (216bp) and peanut specific amplification band (150bp); Extract the DNA in different Walnut Milk materials, utilization is that plant universal primer (CP03-F, CP03-R) carries out pcr amplification to extracted DNA with reference to primer, observe and whether have the amplification of 123bp band, prevent false negative, utilize designed two pairs of primers to carry out the PCR specific amplification of goal gene; Pcr amplification result is analyzed.
The technology of the present invention content comprises:
(1) the method is used soybean, peanut Auele Specific Primer to carry out double PCR detection to different plant milk beverages:
Soybean primer:
Bd 28K-F：5’-GCAGCATTGACCCCTCTACAAGC-3’，
Bd 28K-R：5’-TTCGAATCCAGAAAGCACCGAGT-3’，
Peanut primer:
Ara h1-F：5’-GCCATCAACGCTTCCTCCGAACTCC-3’，
Ara h1-R：5’-TTCACCCGACCCAGGGAATGCT-3’。
In the present invention, in double PCR reaction system, each component composition is as follows:
Composition application of sample amount
2×Premix Taq (TaKaRa) 12.5μl
Primer Bd 28K-F (10 μ M) 0.5 μ l
Primer Bd 28K-R (10 μ M) 0.5 μ l
Primer Ara h1-F (10 μ M) 0.5 μ l
Primer Ara h1-R (10 μ M) 0.5 μ l
DNA sample 1.0 μ l
Rnase Free dH2O 9.5μl
Cumulative volume 25 μ l.
In dual-PCR method of the present invention, amplification program is:
（1）94℃ 10min
（2）94℃ 30s
（3）58℃ 30s
（4）72℃ 30s
(5) get back to the 2nd step, repeat 40 times.
（6）72℃ 7min。
(2) use plant universal primer to carry out PCR detection to different plant milk beverages:
Universal primer:
CP03-F：5'-CGGACGAGAATAAAGATAGAGT-3'
CP03-R：5'-TTTTGGGGATAGAGGGACTTGA-3'
In the present invention, in PCR reaction system, each component composition is as follows:
Composition application of sample amount
2×Premix Taq (TaKaRa) 12.5μl
Primer CP03-F (10 μ M) 0.5 μ l
Primer CP03-F (10 μ M) 0.5 μ l
DNA sample 1.0 μ l
Rnase Free dH2O 10.5μl
Cumulative volume 25 μ l
The general PCR method amplification program of plant is:
（1）94℃ 10min
（2）94℃ 30s
（3）56℃ 30s
（4）72℃ 30s
(5) get back to the 2nd step, repeat 40 times
（6）72℃ 7min
Method of the present invention specifically comprises the following steps:
(1) the soybean gene sequences Design soybean specific PCR primer based on downloading from NCBI:
Bd 28K-F：5’-GCAGCATTGACCCCTCTACAAGC-3’，
Bd 28K-R：5’-TTCGAATCCAGAAAGCACCGAGT-3’；
(2) design of the peanut gene order based on downloading from NCBI peanut specific PCR primer:
Ara h1-F：5’-GCCATCAACGCTTCCTCCGAACTCC-3’，
Ara h1-R：5’-TTCACCCGACCCAGGGAATGCT-3’ ；
(3) By consulting literatures, utilizes plant universal PC R primer:
CP 03-F：5'-CGGACGAGAATAAAGATAGAGT-3'，
CP 03-R：5'-TTTTGGGGATAGAGGGACTTGA-3'；
(4) extract the DNA in Walnut Milk sample, press double PCR reaction system and amplification program, utilize soybean and peanut Auele Specific Primer to carry out double PCR, according to stripe size, judge in Walnut Milk, whether to mix soybean, peanut composition, when amplified band size is 216bp, prove soybean components; When amplified band size is 150bp, prove peanut composition;
(5) DNA extracting from all kinds of Walnut Milk drinks, forms with program and increases with plant universal primer and corresponding PCR reacted constituent, and product stripe size is single while being 123bp, and DNA extraction success be described, meets PCR and reacts requirement.
Present method and common leguminous plants and common nuts, if fibert, Chinese chestnut, walnut, pine nut, Pistacia vera, green fruit, cashew nut, almond, non-nuts are as apple etc. does not have cross reaction, have specificity.Compared with prior art, because protein in product is destroyed, soybean, peanut composition in the food that ELISA method can not effectively detect, soybean, peanut composition that the present invention utilizes double PCR to detect to take Walnut Milk drink that protein ingredient is main raw material to mix simultaneously.The method amplified target gene of multiplex PCR for the present invention, the complex process of having avoided ELISA method to react detects two kinds of compositions simultaneously, saves time; The interference of other albumen that PCR authentication method is subject to is less, more more accurate than ELISA method.
accompanying drawing explanation
Fig. 1 is that soybean of the present invention, peanut double PCR primer are for common fabaceous specificity checking electrophorogram, M:2000bp ladder DNA maker; 1-Semen Glycines; 2-green soybean; 3-Semen sojae atricolor; 4-French beans; 5-mung bean; 6-broad bean; 7-Semen Phaseoli Vulgaris; 8-red kidney bean; 9-pea; 10-fiber crops cowpea; 11-cowpea; 12-Semen Ormosiae Hosiei; 13-red bean; 14-negative control;
Fig. 2 is that soybean of the present invention, peanut double PCR primer are verified electrophorogram, M:2000bp ladder DNA maker for the specificity of other common non-leguminous plants; 1-Semen Glycines; 2-cashew nut; 3-walnut; 4-peanut; 5-oat; 6-banana; 7-white sesame; 8-fibert; Green fruit of 9-; 10-apple; 11-Pistacia vera; 12-melon seeds; 13-Semen Sesami Nigrum; 14-almond; 15-pine nut; 16-negative control;
Fig. 3 is plant universal primer amplification different brands Walnut Milk electrophorogram of the present invention; M:500bp ladder DNA maker; 1 ~ 22: different brands Walnut Milk; 23-negative control;
Fig. 4 is soybean, peanut double PCR primer amplification different brands Walnut Milk electrophorogram; M:2000bp ladder DNA maker; 1 ~ 22: different brands Walnut Milk; 23-negative control.
Embodiment
Below by drawings and Examples, the present invention is described in further detail, but protection domain of the present invention is not limited to described content.
embodiment 1:
1, design of primers
By Literature Consult and BLAST, compare, with reference to NCBI sequence number, be EU493455, EU493457, EU493458, EU493459, EU493460, EU493461, utilize Primer Premier 5.0 and Oligo 6.0 softwares to carry out design of primers, design soybean Auele Specific Primer is synthetic by Hua Da gene biological company limited, and primer sequence is as follows:
Bd 28K-F：5’-GCAGCATTGACCCCTCTACAAGC-3’，
Bd 28K-R：5’-TTCGAATCCAGAAAGCACCGAGT-3’；
By Literature Consult and BLAST, comparing, is AF43223 with reference to NCBI sequence number, AB440237, and AY581852, HM640249, utilizes Primer Premier 5.0 and Oligo6 software to carry out design of primers; The peanut Auele Specific Primer designing is synthetic by Hua Da gene biological company limited, and primer sequence is as follows:
Ara h1-F：5’-GCCATCAACGCTTCCTCCGAACTCC-3’，
Ara h1-R：5’-TTCACCCGACCCAGGGAATGCT-3’。
2, the extraction of DNA
Sample: pulse family class is as Semen Glycines, green soybean, Semen sojae atricolor, French beans, mung bean, broad bean, Semen Phaseoli Vulgaris, red kidney bean, pea, numb cowpea, cowpea, Semen Ormosiae Hosiei, red bean; Nuts is as peanut, pine nut, almond, cashew nut, green fruit, Pistacia vera, fibert, melon seeds, walnut; Non-nuts is as oat, apple, banana, white sesame, Semen Sesami Nigrum.
Get respectively above-mentioned appropriate sample, rapid grind into powder in adding the mortar of liquid nitrogen, weigh respectively 100mg sample powder and be transferred in 1.5mlEP pipe, use the centrifugal column type plant genome DNA of Tian Gen company to extract the extraction that test kit completes DNA in sample, concrete operation step is as follows:
(1) the 700 μ lGP1 that add 0.70ul mercaptoethanol are joined respectively to each containing in the EP pipe of 100mg sample powder, put upside down and mix, 65 ℃ of water-bath 20min;
(2) in each EP pipe, add equal-volume chloroform to mix, precipitate several minutes after layering, centrifugal 5 min of 12000rpm;
(3) upper strata water is proceeded to new centrifuge tube, add 700ul damping fluid GP2 to mix;
(4) mix liquid and add CB3 post, the centrifugal 1min of 12000 rpm, abandons liquid;
(5) in CB3, add 500ul GD liquid, the centrifugal 1min of 12000rpm, abandons liquid, and CB3 post is put into collection tube;
(6) in CB3 post, add 700ul PW liquid, the centrifugal 1min of 12000rpm, abandons liquid, and CB3 post is put into collection tube;
(7) to CB3 post, add 500ul PW liquid, the centrifugal 1min of 12000rpm, abandons liquid;
(8) CB3 post is put back to collection tube, the centrifugal 2min of 12000rpm, abandons liquid, and CB3 post is placed in room temperature placement and dries;
(9) CB3 post proceeds in new centrifuge tube, adds 50ul TE elutriant, and room temperature is placed 5min, the centrifugal 2min of 12000rpm;
(10) the collection liquid the 9th step being obtained adds in CB3 post again, the centrifugal 2min of 12000rpm;
(11) product is stored in to-80 ℃, stand-by.
3, pcr amplification
(1) DNA sample that uses soybean, peanut primer pair to extract carries out double PCR detection, and in PCR reaction system, each component composition is as follows:
In the present invention, in double PCR reaction system, each component composition is as follows:
Composition application of sample amount
2×Premix Taq (TaKaRa) 12.5μl
Primer Bd 28K-F (10 μ M) 0.5 μ l
Primer Bd 28K-R (10 μ M) 0.5 μ l
Primer Ara h1-F (10 μ M) 0.5 μ l
Primer Ara h1-R (10 μ M) 0.5 μ l
DNA sample 1.0 μ l
Rnase Free dH
2O 9.5μl
Cumulative volume 25 μ l;
In dual-PCR method, amplification program is:
（1）94℃ 10min
（2）94℃ 30s
（3）58℃ 30s
（4）72℃ 30s
(5) get back to the 2nd step, repeat 40 times.
（6）72℃ 7min。
4, electrophoresis detection
Get 6 μ L amplified productions electrophoresis on 1.0% sepharose, use TAE as electrophoretic buffer, electrophoresis 20min under 120V, EB dyeing, observes under ultraviolet lamp and uses gel analysis system to take a picture, as shown in Figure 1, Figure 2.In Fig. 1, only soy bean DNA is that swimming lane 1, swimming lane 2, swimming lane 3 occur that specific band is 216bp, and in Fig. 2, Semen Glycines DNA is that swimming lane 1 occurs that specific band is 216bp, and peanut DNA swimming lane 4 occurs that specific band is 150bp, illustrates that primer specificity is strong.
embodiment 2:
Sample: commercially available 22 kinds of different brands are indicated the Walnut Milk drink containing walnut protein composition, and the place of production is distributed in the ground such as Yunnan Province, Hebei province, Shanxi Province, the Inner Mongol.
1, the extraction of DNA
By the lyophilize of difference Walnut Milk sample to be measured, weigh respectively 100mg sample powder and be transferred in 1.5mlEP pipe, use the centrifugal column type plant genome DNA of Tian Gen company to extract the extraction that test kit completes DNA in sample, concrete operation step is with embodiment 1 step 2.
2, pcr amplification
(1) with plant universal primer and corresponding PCR reacted constituent, form with program extracted DNA sample is increased, product stripe size is single while being 123bp, DNA extraction be described successfully, meets PCR and reacts requirement.
In PCR reaction system, each component composition is as follows:
Composition application of sample amount
2×Premix Taq (TaKaRa) 12.5μl
Primer CP03-F (10 μ M) 0.5 μ l
Primer CP03-F (10 μ M) 0.5 μ l
DNA sample 1.0 μ l
Rnase Free dH2O 10.5μl
Cumulative volume 25 μ l
Plant universal primer PCR method amplification program is:
get back to the 2nd step, repeat 40 times
72℃ 7min
(2) DNA sample that uses soybean and peanut primer pair to extract carries out double PCR detection;
In double PCR reaction system, each component composition is as follows:
Composition application of sample amount
2×Premix Taq (TaKaRa) 12.5μl
Primer Bd 28K-F (10 μ M) 0.5 μ l
Primer Bd 28K-R (10 μ M) 0.5 μ l
Primer Ara h1-F (10 μ M) 0.5 μ l
Primer Ara h1-R (10 μ M) 0.5 μ l
DNA sample 1.0 μ l
Rnase Free dH
2O 9.5μl
Cumulative volume 25 μ l.
In dual-PCR method, amplification program is:
94℃ 30s
72℃ 30s
72℃ 7min。
3, electrophoresis detection
Get 6 μ L amplified productions electrophoresis on 1.0% sepharose, use TAE as electrophoretic buffer, electrophoresis 20min under 120V, EB dyeing, observes under ultraviolet lamp and uses gel analysis system to take a picture, as Fig. 3, Fig. 4.In Fig. 3, except other equal positive band 123bp of negative control (swimming lane 23), illustrate that Walnut Milk DNA extraction successfully.The negative contrast of swimming lane 23 in Fig. 4, there is soybean specific band 216bp in swimming lane 1,22, and there is peanut specific band 150bp in swimming lane 1,5,8,12,14,18,21, and interpret sample 1 contains soybean and peanut composition, meets identification of product; Sample 22 contains soybean components, and identification of product is pure Walnut Milk, and result and identification of product are not inconsistent, and has illustrated that soybean components is adulterated.There is peanut compositional banding in sample 5,8,12,14,18,21, wherein only have sample 5,18,21 to indicate and contain peanut composition, and sample 8,12,14 is not indicated containing peanut composition, illustrated that peanut composition is adulterated, sample 2,3,4,6,7,9,10,11,13,15,16,17,19,20 meets identification of product.
Sequence table
<110> Kunming University of Science and Technology
The dual PCR detection method of soybean, peanut composition in <120> Walnut Milk drink
<160> 6
<170> PatentIn version 3.5
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<211> 23
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<212> DNA
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gccatcaacg cttcctccga actcc 25
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Claims (3)
1. a dual PCR detection method for soybean, peanut composition in Walnut Milk drink, is characterized in that: the DNA of testing sample of take is template, with soybean primer as follows, peanut primer, carries out double PCR augmentation detection; If there is specific amplification band, show to comprise in testing sample soybean or peanut composition;
Soybean primer:
Bd 28K-F：5’-GCAGCATTGACCCCTCTACAAGC-3’，
Bd 28K-R：5’-TTCGAATCCAGAAAGCACCGAGT-3’，
Peanut primer:
Ara h1-F：5’-GCCATCAACGCTTCCTCCGAACTCC-3’，
Ara h1-R：5’-TTCACCCGACCCAGGGAATGCT-3’。
2. the dual PCR detection method of soybean, peanut composition in Walnut Milk drink according to claim 1, is characterized in that: PCR reaction system is as follows:
2×Premix Taq (TaKaRa) 12.5μl
Primer Bd 28K-F (10 μ M) 0.5 μ l
Primer Bd 28K-R (10 μ M) 0.5 μ l
Primer Ara h1-F (10 μ M) 0.5 μ l
Primer Ara h1-R (10 μ M) 0.5 μ l
DNA sample 1.0 μ l
Rnase Free dH
2O 9.5μl
Cumulative volume 25 μ l.
3. the dual PCR detection method of soybean, peanut composition in Walnut Milk drink according to claim 1, is characterized in that: pcr amplification program is: 94 ℃ of 10min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, 40 circulations; 72 ℃ of 7min.
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Cited By (6)
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| CN108103233A (en) * | 2018-02-08 | 2018-06-01 | 苏州百源基因技术有限公司 | For detecting the specific primer of peanut DNA and probe and real-time fluorescence quantitative PCR kit |
| CN109880823A (en) * | 2019-04-04 | 2019-06-14 | 中国计量大学 | The extracting method of Walnut Milk DNA |
| CN109971838A (en) * | 2019-04-04 | 2019-07-05 | 中国计量大学 | The PCR-HRM method for identifying molecules of Walnut Milk true or false |
| CN110438251A (en) * | 2019-05-07 | 2019-11-12 | 广东出入境检验检疫局检验检疫技术中心 | Utilize the method for peanut ingredient in dual digital pcr quantitative detection hazel Rong |
| CN111172311A (en) * | 2020-01-08 | 2020-05-19 | 西北民族大学 | Double-fluorescence quantitative PCR detection kit for soybean and walnut allergen genes |
| JP7446956B2 (en) | 2020-09-02 | 2024-03-11 | 株式会社日清製粉グループ本社 | DNA detection method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103233A (en) * | 2018-02-08 | 2018-06-01 | 苏州百源基因技术有限公司 | For detecting the specific primer of peanut DNA and probe and real-time fluorescence quantitative PCR kit |
| CN109880823A (en) * | 2019-04-04 | 2019-06-14 | 中国计量大学 | The extracting method of Walnut Milk DNA |
| CN109971838A (en) * | 2019-04-04 | 2019-07-05 | 中国计量大学 | The PCR-HRM method for identifying molecules of Walnut Milk true or false |
| CN110438251A (en) * | 2019-05-07 | 2019-11-12 | 广东出入境检验检疫局检验检疫技术中心 | Utilize the method for peanut ingredient in dual digital pcr quantitative detection hazel Rong |
| CN111172311A (en) * | 2020-01-08 | 2020-05-19 | 西北民族大学 | Double-fluorescence quantitative PCR detection kit for soybean and walnut allergen genes |
| CN111172311B (en) * | 2020-01-08 | 2023-06-30 | 西北民族大学 | Dual-fluorescence quantitative PCR detection kit for soybean walnut allergen genes |
| JP7446956B2 (en) | 2020-09-02 | 2024-03-11 | 株式会社日清製粉グループ本社 | DNA detection method |
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