CN102367472A - Single/duplex PCR (polymerase chain reaction) detection method for botanical adding material in milk or milk powder - Google Patents
Single/duplex PCR (polymerase chain reaction) detection method for botanical adding material in milk or milk powder Download PDFInfo
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- CN102367472A CN102367472A CN2011101891654A CN201110189165A CN102367472A CN 102367472 A CN102367472 A CN 102367472A CN 2011101891654 A CN2011101891654 A CN 2011101891654A CN 201110189165 A CN201110189165 A CN 201110189165A CN 102367472 A CN102367472 A CN 102367472A
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Abstract
Belonging to the biotechnological field of food, the invention provides a single/duplex PCR (polymerase chain reaction) detection method for a botanical adding material in milk or milk powder. The reagents adopted by the method comprises Taq enzyme, dNTP (deoxyribonucleoside triphosphate), 10* buffer, MgCl2, sterile water, primers and template DNA. The detection method consists of the steps of: conducting extraction and PCR amplification to the DNA template of a detected sample, and analyzing an amplified product so as to determine whether existing a botanical adding material. With a detection limit of 0.1%, the method of the invention can rapidly and accurately detect a botanical adding material in a tested sample so as to provide an effective solution to existing adulterated detection vulnerability in dairy enterprises, and provides a research foundation for realizing quality control of milk and dairy products on a molecular level. Characterized by low cost, high sensitivity, simple operation and less time consumption, the method provided in the invention is suitable for the food quality and safety detection field.
Description
Technical field
The invention belongs to biological technical field, is a kind of pcr amplification detection method that is applicable to the plant-sourced admixture in milk or the milk powder.
Background technology
Milk is nutritious, and the protein content high quality is excellent, receives liking of human consumer.Milk powder is the dry products of milk, in milk-product, has critical role.But present Market competition, part retailer often mingles for speculating, and admixture plant-sourced admixture comprises soya-bean milk, bean powder, soybean protein isolate etc. in milk and milk preparation.Traditional chemical process that plant constituent in the milk is detected has had research, but detected object is single, sensitivity is low because these methods exist, be subject to shortcoming such as extrinsic factor interference, can't accurately, quick, high-throughout distinguish discriminating.National Standard Method is to protein detection complex equipments then, operates more loaded down with trivial detailsly, and expense is higher, and is consuming time longer.
Summary of the invention
The present invention exists sensitivity low for the chemical process that solves plant-sourced composition detection in milk or the milk powder; Be subject to shortcomings such as extrinsic factor interference; The problem that particularly at present protein measuring method can not make a distinction vegetable-protein and animal proteinum in the food, and then a kind of PCR detection method that is applicable to the plant-sourced admixture in milk or the milk powder is provided.
The present invention solves the scheme that its technical problem adopts:
One weight of plant-sourced admixture/dual PCR detection method in a kind of milk or the milk powder; At first the dna profiling to product to be measured extracts; Adopt Newt opposite sex primer and plant-sourced Auele Specific Primer that the genomic dna that extracts is carried out pcr amplification then; Simultaneously the ox source DNA is carried out pcr amplification as negative control group, the plant source DNA is carried out pcr amplification as positive controls, then all amplified productions are carried out the agarose gel electrophoresis analysis respectively; Electrophoresis band that occurs according to the test product hole at last and positive controls and negative control group are compared, and promptly whether have added the plant-sourced admixture in the decidable product.
Newt opposite sex primer 1 is classified as with the nucleotides sequence that plant Auele Specific Primer 2 has: primer 1-1 5 '-GCC ATA TAC TCT CCT TGG TGA CA-3 '; Primer 1-2 5 '-GTA GGC TTG GGAATA GTA CGA-3 '; Primer 2-1 5 '-AAT CTT CTA CTG GTA CAT GGA C-3 '; Primer 2-2 5 '-TCA TCA TCT TTG GTA AAA TCA AG-3 '.
PCR reaction agents useful for same also comprises Taq enzyme, dNTP, 10 * damping fluid, MgCl
2And aqua sterilisa.
Said plant-sourced comprises soya-bean milk, bean powder, soybean protein isolate or thin rice gruel.
Described PCR primer concentration is 0.1-0.4 μ mol/L.Preferred plan is that described PCR primer concentration is 0.2 μ mol/L.
Said PCR reaction cycle condition is 92～96 ℃ of 3～8min of preparatory sex change; 92～96 ℃ of 20～40s of sex change, 52～60 ℃ of 20～40s that anneal extend 72 ℃ of 30～120s, and 72 ℃ of 3～10min are extended in totally 31～35 circulations at last.Preferred plan is that said PCR reaction cycle condition is 94 ℃ of 4min of preparatory sex change; 94 ℃ of 30s of sex change, the 54 ℃ of 30s that anneal extend 72 ℃ of 60s, and 72 ℃ of 7min are extended in totally 35 circulations at last.
The mass concentration of carrying out the sepharose of agarose gel electrophoresis analysis employing is 1.5～2.0%.The preferred plan mass concentration is 1.7%.
The present invention adopts the single and double PCR technology in the molecular biology; Can be fast, accurately, from the gene angle situation of mixing of plant-sourced composition milk and the milk powder is carried out qualitative and semi-quantitative analysis delicately, for market surveillance department and inspection and quarantine department provide technical guarantee.This method is simple to operate, and expense is low, weak point consuming time, and specificity is good, and is highly sensitive, and good reproducibility can detect the plant constituent that mixes in the milk and be low to moderate 0.1%, is suitable for applying.
Embodiment
Below set forth in detail the preferred embodiment of the invention.
Embodiment one:
The said detection method of present embodiment may further comprise the steps successively:
A. the dna profiling to product to be measured extracts, and, extracts the DNA of product gene group that is.
The DNA extraction method of product can be any one known method of the prior art; Such as CTAB method, SDS method; Also can be the applicant in application on April 1st, 2011, application number is 201110081615.8, name is called the method for being set forth in " a kind of soybean protein isolate base is because of the process for extracting of group DNA ", difference only is to replace the soybean protein isolate in the above-mentioned application with milk of the present invention or milk powder.The applicant sets forth this method as follows at this:
At first; The milk powder to be measured and TE damping fluid (the concentration is not limit) thorough mixing that will add known quantity bean powder (addition is set forth) are in advance at the back processed TE mixed solution (blending ratio need not to limit); Said TE damping fluid also can replace by water, and purpose only is to make it become liquid;
Then, add the long-pending guanidinium isothiocyanate lysate of liquid diploid, abundant mixing, the room temperature cracking, the cracking time is 20～60min.Contain 50～100mM pH, 6.0～8.0Tris-HCl in the said guanidinium isothiocyanate lysate; 10～100mM pH 8.0EDTA; The 5M guanidinium isothiocyanate; Volume content 1.3%TritonX-100.
The 3rd step: adopt phenol, chloroform/primary isoamyl alcohol extracting albumen, the volume ratio of described phenol, chloroform/primary isoamyl alcohol is 25: 24: 1, and the volume ratio of described chloroform/primary isoamyl alcohol is 24: 1.
The 4th step: adopt isopropanol precipitating DNA, precipitation temperature is-20～30 ℃, and the ST is 20～60min.
The 5th step: washing with alcohol, the ethanol volumetric concentration of use is 70～95%;
The 6th step: room temperature is dried, and the air dried time is 10～15min.
The 7th step: the 6th step products obtained therefrom is dissolved in TE damping fluid (concentration is not limit) or the aqua sterilisa fully, promptly obtains the TE solution of genomic dna, 4 ℃ or-20 ℃ of preservations are subsequent use.
B. the TE solution (being template DNA solution) that aforementioned process is obtained genomic dna carries out pcr amplification, simultaneously ox source DNA and plant source DNA is carried out pcr amplification.The plant-sourced that this patent is mentioned can be soya-bean milk, bean powder, soybean protein isolate, thin rice gruel or other all plant-sourced materials.
The pcr amplification agents useful for same comprises Taq enzyme, dNTP (deoxyribonucleoside triphosphate), 10 * damping fluid, MgCl
2, aqua sterilisa, primer and template DNA.
Present embodiment is the double PCR reaction system, and the processing parameter of control is:
The genomic dna that extracts is carried out pcr amplification adopt Newt opposite sex primer and plant-sourced Auele Specific Primer, described PCR primer concentration is 0.1-0.4 μ mol/L, and preferable primer concentration is 0.2 μ mol/L.Newt opposite sex primer 1 is classified as with the nucleotides sequence that plant Auele Specific Primer 2 has: primer 1-1 5 '-GCCATA TAC TCT CCT TGG TGA CA-3 '; Primer 1-2 5 '-GTA GGC TTG GGA ATAGTA CGA-3 '; Primer 2-1 5 '-AAT CTT CTA CTG GTA CAT GGA C-3 '; Primer 2-25 '-TCA TCA TCT TTG GTA AAA TCA AG-3 '.
The ox source DNA is carried out pcr amplification as negative control group, the plant source DNA is carried out pcr amplification as positive controls, to judge the positive or negative of product plant-sourced admixture to be measured.
Genomic dna, ox source DNA and the plant source DNA of aforementioned extraction carried out pcr amplification all adopt identical temperature and time, that is, cycling condition is 92 ℃ of 3min of preparatory sex change; 92 ℃ of 20s of sex change, the 52 ℃ of 20s that anneal extend 72 ℃ of 30s, and 72 ℃ of 3min are extended in totally 35 circulations at last.
C. all amplified productions are carried out the agarose gel electrophoresis analysis respectively, this electrophoresis analytical method is a known technology.
The mass concentration of carrying out the sepharose of agarose gel electrophoresis analysis employing is 1.5%.
Electrophoresis band that occurs according to the test product hole at last and positive controls and negative control group are compared, i.e. the positive or negative of decidable product plant-sourced to be measured admixture, thus can know whether added the plant-sourced admixture in the product.
The bean powder mass ratio that present embodiment adds accounts for 0.1%, 0.5%, 2.5% of milk powder respectively; 10%, 40%, be product to be measured with this milk powder then; Adopt described technological process of present embodiment and controlled variable; The applicant successfully detects the bean powder in this serial milk powder, and minimum detectability is 0.1%, and its accuracy of detection has reached 100%.
Embodiment two:
The difference of present embodiment and embodiment one only is: be the plant-sourced admixture with the soybean protein isolate, the mass ratio of the shared milk powder of soybean protein isolate still is 0.1%, 0.5%, 2.5%, 3.5%, 5%.With this milk powder is product to be measured, and institute's control process parameters and embodiment one are identical.Conclusion: the applicant successfully detects the soybean protein isolate in this serial milk powder, and minimum detectability is 0.1%, and its accuracy of detection has reached 100%.
Embodiment three:
The difference of present embodiment and embodiment one is:
Among the step a, be the plant-sourced admixture with the soya-bean milk.
Among the step b, the reaction conditions that genomic dna, ox source DNA and the plant source DNA that extracts is carried out the pcr amplification employing is 94 ℃ of 4min of preparatory sex change; 94 ℃ of 30s of sex change, the 54 ℃ of 30s that anneal extend 72 ℃ of 60s, and 72 ℃ of 7min are extended in totally 35 circulations at last.
Among the step c, the mass concentration of carrying out the sepharose of agarose gel electrophoresis analysis employing is 1.7%.
Present embodiment still is the double PCR reaction system, and the processing parameter of control is:
Present embodiment is respectively 0.1%, 0.5%, 2.5% to mix volume(tric)fraction; 10%; The milk of 40% soya-bean milk is product to be measured, adopts described technological process of present embodiment and controlled variable, and the applicant successfully detects the soya-bean milk that contains in this serial milk; Minimum detectability is 0.1%, and its accuracy of detection has reached 100%.
Embodiment four:
The difference of present embodiment and embodiment one only is:
Among the step a, be the plant-sourced admixture with the soya-bean milk.
Among the step b, the reaction conditions that genomic dna, ox source DNA and the plant source DNA that extracts is carried out the pcr amplification employing is 96 ℃ of 8min of preparatory sex change; 96 ℃ of 40s of sex change, the 60 ℃ of 40s that anneal extend 72 ℃ of 120s, and 72 ℃ of 10min are extended in totally 31 circulations at last.
Among the step c, the mass concentration of carrying out the sepharose of agarose gel electrophoresis analysis employing is 2.0%.
Present embodiment adopts substance PCR reaction system, and the processing parameter of control is:
Present embodiment is respectively 0.1%, 0.5%, 2.5% to mix volume(tric)fraction; 10%; The milk of 40% soya-bean milk is product to be measured, adopts described technological process of present embodiment and controlled variable, and the applicant successfully detects the soya-bean milk that contains in this serial milk; Minimum detectability is 0.1%, and its accuracy of detection has reached 100%.
Embodiment five:
The difference of present embodiment and embodiment four only is:
Among the step a, be the plant-sourced admixture with the bean powder.
Among the step c, adopt substance PCR reaction system, the processing parameter of control is:
Present embodiment is to have added the quality of milk powder ratio 0.1%, 0.5%, 2.5% that accounts for respectively; The milk powder of 5%, 10%, 40% bean powder is product to be measured; Adopt described technological process of present embodiment and controlled variable; The applicant successfully detects the bean powder that contains in this serial milk powder, and minimum detectability is 0.1%, and its accuracy of detection has reached 100%.
Sequence table
< 110>Harbin Institute of Technology
< 120>the one weight/dual PCR detection method of plant-sourced admixture in a kind of milk or the milk powder
<160>4
<210>1
<211>23
<212>DNA
< 213>artificial sequence
<220>
< 223>the primer 1-1 of Newt opposite sex primer 1
<400>1
GCCATATACT?CTCCTTGGTG?ACA?23
<210>2
<211>21
<212>DNA
< 213>artificial sequence
<220>
< 223>the primer 1-2 of Newt opposite sex primer 1
<400>2
GTAGGCTTGG?GAATAGTACG?A?21
<210>3
<211>22
<212>DNA
< 213>artificial sequence
<220>
< 223>primer 2 of plant Auele Specific Primer 2-1
<400>3
AATCTTCTAC?TGGTACATGG?AC?22
<210>4
<211>23
<212>DNA
< 213>artificial sequence
<220>
< 223>primer 2 of plant Auele Specific Primer 2-2
<400>4
TCATCATCTT?TGGTAAAATC?AAG?23
Claims (10)
1. the one weight/dual PCR detection method of plant-sourced admixture in milk or the milk powder; It is characterized in that; At first the dna profiling to product to be measured extracts, and adopts Newt opposite sex primer and plant-sourced Auele Specific Primer that the genomic dna that extracts is carried out pcr amplification then, simultaneously the ox source DNA is carried out pcr amplification as negative control group; The plant source DNA is carried out pcr amplification as positive controls, then all amplified productions are carried out the agarose gel electrophoresis analysis respectively; Electrophoresis band that occurs according to the test product hole at last and positive controls and negative control group are compared, and promptly whether have added the plant-sourced admixture in the decidable product.
2. the one weight/dual PCR detection method of plant-sourced admixture in milk according to claim 1 or the milk powder; It is characterized in that Newt opposite sex primer 1 is classified as with the nucleotides sequence that plant Auele Specific Primer 2 has: primer 1-1 5 '-GCC ATATAC TCT CCT TGG TGA CA-3 '; Primer 1-2 5 '-GTA GGC TTG GGA ATA GTA CGA-3 '; Primer 2-1 5 '-AAT CTT CTA CTG GTA CAT GGA C-3 '; Primer 2-2 5 '-TCA TCA TCT TTG GTA AAATCA AG-3 '.
3. the one weight/dual PCR detection method of plant-sourced admixture is characterized in that in milk according to claim 1 or the milk powder, and PCR reaction agents useful for same also comprises Taq enzyme, dNTP, 10 * damping fluid, MgCl
2And aqua sterilisa.
4. the one weight/dual PCR detection method of plant-sourced admixture is characterized in that in milk according to claim 1 or the milk powder, and said plant-sourced comprises soya-bean milk, bean powder, soybean protein isolate or thin rice gruel.
5. according to the one weight/dual PCR detection method of plant-sourced admixture in any described milk of claim 1-4 or the milk powder, it is characterized in that described PCR primer concentration is 0.1-0.4 μ mol/L.
6. the one weight/dual PCR detection method of plant-sourced admixture is characterized in that in milk according to claim 5 or the milk powder, and described PCR primer concentration is 0.2 μ mol/L.
7. according to the one weight/dual PCR detection method of plant-sourced admixture in any described milk of claim 1-4 or the milk powder, it is characterized in that said PCR reaction cycle condition is 92～96 ℃ of 3～8min of preparatory sex change; 92～96 ℃ of 20～40s of sex change, 52～60 ℃ of 20～40s that anneal extend 72 ℃ of 30～120s, and 72 ℃ of 3～10min are extended in totally 31～35 circulations at last.
8. the one weight/dual PCR detection method of plant-sourced admixture is characterized in that in milk according to claim 7 or the milk powder, and said PCR reaction cycle condition is 94 ℃ of 4min of preparatory sex change; 94 ℃ of 30s of sex change, the 54 ℃ of 30s that anneal extend 72 ℃ of 60s, and 72 ℃ of 7min are extended in totally 35 circulations at last.
9. according to the one weight/dual PCR detection method of plant-sourced admixture in any described milk of claim 1-4 or the milk powder, it is characterized in that the mass concentration of carrying out the sepharose of agarose gel electrophoresis analysis employing is 1.5～2.0%.
10. the one weight/dual PCR detection method of plant-sourced admixture is characterized in that in milk according to claim 9 or the milk powder, and the mass concentration of carrying out the sepharose of agarose gel electrophoresis analysis employing is 1.7%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011101891654A CN102367472A (en) | 2011-07-07 | 2011-07-07 | Single/duplex PCR (polymerase chain reaction) detection method for botanical adding material in milk or milk powder |
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| CN2011101891654A CN102367472A (en) | 2011-07-07 | 2011-07-07 | Single/duplex PCR (polymerase chain reaction) detection method for botanical adding material in milk or milk powder |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103103265A (en) * | 2013-01-16 | 2013-05-15 | 北京出入境检验检疫局检验检疫技术中心 | Kit and primers for detecting allergen milk |
| CN103614469A (en) * | 2013-11-20 | 2014-03-05 | 南通市产品质量监督检验所 | Method for identifying components of plant origin protein in raw milk and milk |
| CN107167508A (en) * | 2017-07-13 | 2017-09-15 | 上海交通大学 | The method that non-lactoprotein in cow's milk is detected based on gel electrophoresis and Chemical Measurement |
-
2011
- 2011-07-07 CN CN2011101891654A patent/CN102367472A/en active Pending
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| 岳巧云等: "实时荧光PCR在鉴别奶粉中掺入大豆成分的应用研究", 《食品科学》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103265A (en) * | 2013-01-16 | 2013-05-15 | 北京出入境检验检疫局检验检疫技术中心 | Kit and primers for detecting allergen milk |
| CN103103265B (en) * | 2013-01-16 | 2014-07-30 | 北京出入境检验检疫局检验检疫技术中心 | Kit and primers for detecting allergen milk |
| CN103614469A (en) * | 2013-11-20 | 2014-03-05 | 南通市产品质量监督检验所 | Method for identifying components of plant origin protein in raw milk and milk |
| CN103614469B (en) * | 2013-11-20 | 2016-03-23 | 南通市产品质量监督检验所 | Plant source protein composition discrimination method in a kind of raw dairy and milk |
| CN107167508A (en) * | 2017-07-13 | 2017-09-15 | 上海交通大学 | The method that non-lactoprotein in cow's milk is detected based on gel electrophoresis and Chemical Measurement |
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Application publication date: 20120307 |