CN107365857A - Based on DNA gel electrophoretogram and mtDNA:The method that nDNA values differentiate fresh milk and reconstituted milk - Google Patents
Based on DNA gel electrophoretogram and mtDNA:The method that nDNA values differentiate fresh milk and reconstituted milk Download PDFInfo
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- CN107365857A CN107365857A CN201710708129.1A CN201710708129A CN107365857A CN 107365857 A CN107365857 A CN 107365857A CN 201710708129 A CN201710708129 A CN 201710708129A CN 107365857 A CN107365857 A CN 107365857A
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- milk
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
Abstract
The invention discloses one kind to be based on DNA gel electrophoretogram and mtDNA:The method that nDNA values differentiate fresh milk and reconstituted milk, this method extract cow genome group DNA first from milk sample to be measured, then enter row agarose gel electrophoresis analysis to the DNA of extraction, and initial characterization is carried out to testing sample according to the DNA bands extracted.Then Real-Time Fluorescent Quantitative PCR Technique is recycled, while expands the cow genome group DNA of extraction mitochondrial DNA (mtDNA) and core DNA (nDNA), and then calculates mtDNA:NDNA values, it may further determine that testing sample is fresh milk or reconstituted milk according to the size of the value.The present invention provides a kind of new method for the detection of milk quality with evaluation, and also producing high-quality dairy products for enterprise provides reference, has a extensive future, and the nutritional quality detection for milk has great importance.
Description
Technical field
The invention belongs to food quality detection technical field, and in particular to a kind of DNA gel electrophoretogram and mtDNA:nDNA
The method that value differentiates new fresh milk and recovery milk.
Background technology
In recent years, milk and milk productses quality and safety problem emerge in an endless stream, and are increasingly paid close attention to by consumer.It is raw
The dairy products of output high-quality be unable to do without high-quality raw material, thereby, it is ensured that the quality of raw material is just particularly important.State at present
Interior dairy produce consumption is mainly UHT milk, and national standard clear stipulaties, the raw materials for production of UHT milk can be fresh milk or recovery
Milk, but the raw material title used must be clearly marked in commodity packaging, but there still have the illegal businessman of small part to use to be multiple
Do not mark clearly, the rights and interests of serious infringement consumer, therefore establish a kind of reliable and stable fresh after former milk production UHT milk
Breast and recovery milk discrimination method are particularly important.
The most important method of detection recovery milk is the content of high effective liquid chromatography for measuring wherein chaff propylhomoser at present, so as to
Identify recovery milk, clear stipulaties in the standard that the Ministry of Agriculture of China issues：Chaff propylhomoser in every 100 grams of protein in pasturising milk
Content is more than 12 milligram hours, it is possible to determine that contains recovery milk in the product.But only judged using this index, often
It is possible that the result of false positive, such as dairy produce furosine level during storage may increase, therefore establish new
Recovery milk discrimination method with regard to imperative.
In recent years, the powerful that Protocols in Molecular Biology has turned into dairy products quality testing and molecular nutrition is studied.Profit
The identification that milk quality is carried out with the method for molecular biology has become a kind of trend.It is well known that DNA is a kind of thermal sensitivity
Large biological molecule, food in process often through heating, this will affect to DNA.Than
Such as milk powder is exactly that milk produces by pasteurization, homogeneous, spray drying series of process, by adding certain ratio
The water of example reconciles into recovery milk, and this has resulted in the difference of new fresh milk and recovery milk in DNA qualities.
The content of the invention
The defects of technical problems to be solved by the invention are to differentiate new fresh milk and recovery milk for prior art and not
Foot, there is provided a kind of detection efficiency is high, it is practical, based on DNA gel electrophoretogram and mtDNA:NDNA value differences are different fresh to identify
The method of breast and recovery milk.
Technical scheme comprises the steps of used by solving above-mentioned technical problem：
1st, cow genome group DNA is extracted from testing sample.
2nd, the specific primer of the 12S rRNA genes on ox mitochondrial DNA is designed and synthesized：
Sense primer：5’-CGC GGT CAT ACG ATT AAC CC-3’
Anti-sense primer：5’-AAC CCT ATT TGG TAT GGT GCT T-3’
3rd, the specific primer of the β-globin genes on ox core DNA is designed and synthesized：
Sense primer：5’-CGG CGG CGG GCG GCG CGG GCT GGG CGG GAA GGC CCA TGG CAA
GAA GG-3’
Anti-sense primer：5’-GCC GGC CCG CCG CGC CCG TCC CGC CGC TCA CTC AGC GCA GCA
AAG G-3’
4th, the cow genome group DNA extracted to step 1 enters row agarose gel electrophoresis, according to the difference of respective electrophoretic band,
Initial characterization is carried out to testing sample：DNA agarose gel electrophoresis figure bands are bright and without hangover and diffusing phenomenon, illustrate to treat test sample
Product are fresh milks, and DNA agarose gel electrophoresis figures band is slightly dark and hangover and diffusing phenomenon are serious, and it is multiple to illustrate testing sample
Aurochs milk.
5th, the cow genome group DNA that step 1 is extracted is used to be carried out respectively to the specific primer of step (2) and (3) for template
Real-time fluorescence quantitative PCR expands, while expands 12S rRNA genes and β-globin genes, respective Ct values is obtained, according to public affairs
Formula mtDNA:NDNA=2-(Ct12S rRNA-Ctβ-globin)Calculate mtDNA:NDNA values, work as mtDNA:NDNA values are 20~100, are said
Bright testing sample is fresh milk, works as mtDNA:NDNA values are 120~2000, and it is reconstituted milk to illustrate testing sample.
Testing result with reference to above-mentioned steps 4 and 5 is that the accurate discriminating of fresh milk and reconstituted milk can be achieved.
In above-mentioned steps 5, the condition of the PCR amplifications is：95 DEG C of pre-degeneration 10min；95 DEG C of denaturation 30s, 60 DEG C of annealing
1min, 72 DEG C of extension 1min, 40 circulations.
The present invention finally sets up fresh milk and answered by comparing DNA quality discrepancies in fresh milk and reconstituted milk
The molecular biology method that aurochs milk differentiates, i.e., cow genome group DNA is extracted from testing sample respectively first, then to extraction
DNA enters row agarose gel electrophoresis analysis, and initial characterization is carried out to testing sample according to the DNA bands extracted.Then it is sharp again
With Real-Time Fluorescent Quantitative PCR Technique, while expand the cow genome group DNA of extraction mitochondrial DNA (mtDNA) and core DNA
(nDNA), and then mtDNA is calculated:NDNA values, it may further determine that testing sample is fresh milk according to the size of the value
Or reconstituted milk.
Compared with prior art, present invention firstly discovers that fresh milk and reconstituted milk in DNA bands integralities and
mtDNA:Difference in nDNA values, provide new thinking to distinguish fresh milk and reconstituted milk, it is possible to achieve to extensive
Fresh milk carry out preliminary judgement, be advantageous to market monitoring, have a extensive future, the monitoring for milk quality has important
Meaning.
Brief description of the drawings
Fig. 1 is the DNA extracted in fresh milk and reconstituted milk agarose gel electrophoresis figure.
Embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but protection scope of the present invention is not limited only to
These embodiments.
Embodiment 1
1st, fresh milk sample collection is in the milk cow of Xian District, Shanxi Province suburban area raiser, the collection capacity of each sample
40mL, it is respectively charged into 50mL centrifuge tubes, using analysis of milk composition instrument to milk constituents (butterfat, lactoprotein, lactose, total solid
Thing content) directly detected；The fresh milk of collection is divided into four groups, within first group of normal temperature preserves 24 hours, second group
Within Cord blood 48 hours, the 3rd group of pasteurize 30min at 65 DEG C, the 4th group of pasteurize 15s at 5 DEG C.
Whole-fat milk powder is purchased from Xi'an China Resources Wan Jia supermarkets, according to the solid component content of fresh milk by whole-fat milk powder
Restored, be specially：Weigh 120g whole-fat milk powders and add in 800mL distilled water and dissolve, being then settled to 1L with distilled water holds
Measuring bottle, obtain the reconstituted milk that milk powder content is 12%.
Above-mentioned fresh milk and reconstituted milk are extracted for DNA, specific extracting method is as follows：
Milk sample at 7500rpm, 4 DEG C is centrifuged into 10min in ultrahigh speed refrigerated centrifuge, centrifugation is scraped off with small spoon
The butter fat on pipe upper strata, the milk proem in intermediate layer is removed with suction pipe, leave one layer of precipitation of bottommost, then add into centrifuge tube
Enter 600 μ L pH=7.4 PBS, bottom precipitation is blown and beaten and suspends and is transferred in 1.5mL centrifuge tubes, 12000rpm, 4
DEG C centrifugation 10min, discards supernatant liquid, retains bottom precipitation, then add 60 μ L emulsifying agents into centrifuge tube and (be by mass fraction
The 90% Triton-X100 aqueous solution, the ethanol water that volume fraction is 95%, 0.9g/L the NaCl aqueous solution by volume
For 20:125:855 are formulated) and 540 μ L pH=7.4 PBS, with oscillator vibrate to precipitation completely suspend,
40 DEG C of water bath with thermostatic control processing 10min slough the butter fat around body cell, 12000rpm, 4 DEG C of centrifugation 10min, abandon supernatant, add
500 μ L pH=7.4 PBS, which suspends, to be precipitated, and is made body cell precipitation enrichment in 12000rpm, 4 DEG C of centrifugation 10min, is abandoned
Clear liquid.To body cell precipitate in add 350 μ L DNA Extraction buffers (NaCl 1mol/L, Tris-HCl 0.5mol/L,
EDTA-Na 0.5mol/L, pH are adjusted to 7.5~8.0), 50 μ L SDS lysates (it is double that 20g lauryl sodium sulfate is added into 80mL
Steam water in, in 68 DEG C dissolve by heating after, be adjusted to pH=7.2 with dense HCl, 100mL be settled to distilled water), 10 μ L Proteinase Ks,
Water-bath digests 4 hours at 56 DEG C.Isometric Tris saturation phenol is added into digestion product, centrifugation obtains supernatant；Will
Supernatant is transferred in another 1.5mL centrifuge tube, and it is 25 to add isometric phenol with chloroform, isoamyl alcohol volume ratio:24:1
Mixture, overturn back and forth, dissolve precipitation, 12000rpm centrifuges to obtain supernatant；Supernatant is transferred to another 1.5mL
In centrifuge tube, (- 20 DEG C) precipitations of ice absolute ethyl alcohol are added, are gently shaken, -20 DEG C of standings, 12000rpm centrifugation 10min, discarded
Upper strata ethanol, bottom precipitation are washed with ice absolute ethyl alcohol (- 20 DEG C), 12000rpm, 4 DEG C of centrifugation 10min, careful removal upper strata second
Alcohol, quickly volatilize ethanol, add 25 μ L TE (by 2mL pH=8.0 1mol/L Tris-HCl buffer solutions and
After the 0.4mL0.5mol/L EDTA aqueous solution is well mixed, add distilled water to be settled to 200mL, adjust pH to 8.0 to be formulated) dissolving
Cow genome group DNA, -4 DEG C save backup.
2nd, specific primer is designed and synthesized according to the 12S rRNA gene orders on ox mitochondrial DNA：
Sense primer：5’-CGC GGT CAT ACG ATT AAC CC-3’
Anti-sense primer：5’-AAC CCT ATT TGG TAT GGT GCT T-3’
3rd, specific primer is designed and synthesized according to the β-globin gene orders on ox core DNA：
Sense primer：5’-CGG CGG CGG GCG GCG CGG GCT GGG CGG GAA GGC CCA TGG CAA
GAA GG-3’
Anti-sense primer：5’-GCC GGC CCG CCG CGC CCG TCC CGC CGC TCA CTC AGC GCA GCA
AAG G-3’
4th, the cow genome group DNA for extracting above-mentioned steps 1 from fresh milk and reconstituted milk carries out Ago-Gel electricity
Swimming analysis, as shown in figure 1, extracted from fresh milk obtained DNA agarose gel electrophoresis figure bands it is bright while without hangover and
Diffusing phenomenon, and extract that obtained DNA agarose gel electrophoresis figures band is slightly dark and hangover and diffusing phenomenon from reconstituted milk
Seriously, it can realize that the initial characterization of fresh milk and reconstituted milk differentiates by the difference of the two.
5th, use step 1 extract fresh milk and reconstituted milk cow genome group DNA for template, respectively to step 2,3
Specific primer carry out real-time fluorescence quantitative PCR amplification, PCR amplification reaction system be：Each 0.4 μ L of upstream and downstream primer,
The μ L of 1 μ L, 2 × Ultal SYBR Mixture of the DNA profiling 5 and μ L of distilled water 3.2；PCR amplification conditions are：95 DEG C of pre-degenerations
10min；95 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72 DEG C of extension 1min, 40 circulate.
Each self-corresponding 12S of both fresh milk and reconstituted milk can be obtained by real-time fluorescence quantitative PCR amplification
The Ct values of rRNA genes and β-globin genes, then according to formula mtDNA:NDNA=2-(Ct12S rRNA-Ctβ-globin), wherein
Ct12S rRNAThe fluorescence signal for representing 12S rRNA genes reaches the period undergone during the thresholding of setting, Ctβ-globinRepresent
The fluorescence signal of β-globin genes reaches the period undergone during the thresholding of setting, can calculate fresh milk and recovery
The respective mtDNA of milk:NDNA values, it can be found that the mtDNA of reconstituted milk:NDNA values are significantly higher than fresh milk, fresh ox
The mtDNA of milk:NDNA values are 20~100, the mtDNA of recovery milk:NDNA values are 120~2000.Therefore according to therebetween
Difference can further realize the quantitative discriminating of fresh milk and reconstituted milk.
Testing result with reference to above-mentioned steps 4 and 5 is that the accurate discriminating of fresh milk and reconstituted milk can be achieved.
Claims (2)
1. one kind is based on DNA gel electrophoretogram and mtDNA:NDNA values differentiate the method for fresh milk and reconstituted milk, its feature
It is that it is comprised the steps of：
(1) cow genome group DNA is extracted from testing sample；
(2) specific primer of the 12S rRNA genes on ox mitochondrial DNA is designed and synthesized：
Sense primer：5’-CGC GGT CAT ACG ATT AAC CC-3’
Anti-sense primer：5’-AAC CCT ATT TGG TAT GGT GCT T-3’
(3) specific primer of the β-globin genes on ox core DNA is designed and synthesized：
Sense primer：5’-CGG CGG CGG GCG GCG CGG GCT GGG CGG GAA GGC CCA TGG CAA GAA
GG-3’
Anti-sense primer：5’-GCC GGC CCG CCG CGC CCG TCC CGC CGC TCA CTC AGC GCA GCA AAG
G-3’
(4) row agarose gel electrophoresis are entered to the cow genome group DNA of step (1) extraction, it is right according to the difference of respective electrophoretic band
Testing sample carries out initial characterization：DNA agarose gel electrophoresis figure bands are bright and without hangover and diffusing phenomenon, illustrate testing sample
It is fresh milk, DNA agarose gel electrophoresis figures band is slightly dark and hangover and diffusing phenomenon are serious, illustrates testing sample to restore
Milk；
(5) the cow genome group DNA that step (1) is extracted is used to be carried out respectively to the specific primer of step (2) and (3) for template
Real-time fluorescence quantitative PCR expands, while expands 12S rRNA genes and β-globin genes, respective Ct values is obtained, according to public affairs
Formula mtDNA:NDNA=2-(Ct12S rRNA-Ctβ-globin)Calculate mtDNA:NDNA values, work as mtDNA:NDNA values are 20~100, are said
Bright testing sample is fresh milk, works as mtDNA:NDNA values are 120~2000, and it is reconstituted milk to illustrate testing sample；
It is that the accurate discriminating of fresh milk and reconstituted milk can be achieved with reference to above-mentioned steps (4) and the testing result of (5).
2. according to claim 1 be based on DNA gel electrophoretogram and mtDNA:NDNA values differentiate fresh milk and restore ox
The method of milk, it is characterised in that：In step (5), the condition of the PCR amplifications is：95 DEG C of pre-degeneration 10min；95 DEG C of denaturation
30s, 60 DEG C of annealing 1min, 72 DEG C of extension 1min, 40 circulate.
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| CN201710708129.1A CN107365857B (en) | 2017-08-17 | 2017-08-17 | Method for identifying fresh milk and recovered milk based on DNA gel electrophoresis chart and mtDNA (deoxyribonucleic acid). nDNA value |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719426A (en) * | 2012-06-08 | 2012-10-10 | 陕西师范大学 | Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification |
| CN105018606A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality based method for evaluation of milk freshness |
| CN105445393A (en) * | 2015-11-17 | 2016-03-30 | 中国农业科学院北京畜牧兽医研究所 | Identification method for reconstituted milk in pasteurized milk |
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2017
- 2017-08-17 CN CN201710708129.1A patent/CN107365857B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719426A (en) * | 2012-06-08 | 2012-10-10 | 陕西师范大学 | Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification |
| CN105018606A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality based method for evaluation of milk freshness |
| CN105445393A (en) * | 2015-11-17 | 2016-03-30 | 中国农业科学院北京畜牧兽医研究所 | Identification method for reconstituted milk in pasteurized milk |
Non-Patent Citations (4)
| Title |
|---|
| J. LIAO等: "Development of a rapid mitochondrial DNA extraction method for species identification in milk and milk products", 《J. DAIRY SCI》 * |
| L. LAUBENTHAL等: "Mitochondrial DNA copy number and biogenesis in different tissues of early-and late-lactating dairy cows", 《JOURNAL OF DAIRY SCIENCE》 * |
| MAURO MUSTO等: "Changes of DNA quality and meat physicochemical properties in bovine supraspinatus muscle during microwave heating", 《JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE》 * |
| 王荣泰等: "《美食百科》", 31 July 2015, 新华出版社 * |
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