CN101665825A - Method for identifying falsification of distilled spirit by utilizing nucleic acid detection technique - Google Patents
Method for identifying falsification of distilled spirit by utilizing nucleic acid detection technique Download PDFInfo
- Publication number
- CN101665825A CN101665825A CN200910035889A CN200910035889A CN101665825A CN 101665825 A CN101665825 A CN 101665825A CN 200910035889 A CN200910035889 A CN 200910035889A CN 200910035889 A CN200910035889 A CN 200910035889A CN 101665825 A CN101665825 A CN 101665825A
- Authority
- CN
- China
- Prior art keywords
- dna
- distilled spirit
- liquor
- falsification
- wine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for identifying falsification of distilled spirit by utilizing a nucleic acid detection technique, belonging to the biological high-technology field and comprising the following steps: adding a DNA solution to distilled spirit, and then extracting DNA added to the distilled spirit, adopting PCR to react so as to detect a target gene, and then judging the falsification of the distilled spirit according to the amount of amplified products in an electrophoresis result. The method has the characteristics of rapidness, simplicity, accuracy, strong specificity and high sensitivity. The method is suitable for providing technology support to each distilled spirit manufacturer, quality monitoring departments at various levels and market supervision departments to enable the manufacturers and the departments actively, rapidly and accurately take action in safeguarding legal rights and executing the law so as to protect the lawful rights and interests of enterprises and consumers.
Description
One, technical field
The present invention relates to a kind of method of utilizing nucleic acid detection technique to differentiate falsification of distilled spirit, belong to biological high-tech field.Have very important significance aspect false proof at liquor.
Two, background technology
Liquor is the exclusive traditional product of China, and is with a long history.In the last few years, especially the demand to famous-brand and high-quality goods was more and more big to the demand of liquor, how to differentiate that true and false famous-brand and high-quality liquor has been subjected to people's extensive attention.The false proof main employing of liquor at present locates to use anti-counterfeit package and anti-counterfeiting printing technology in bottleneck, bottle cap, the outer packaging of alcohol product, identify, seal etc.Though certain anti-fake effect is played in these false proof measures, wish with the public's psychology and the demand of market management all also has certain distance.They itself have remarkable antiforge function, perhaps also have perfect phone, network system as support, but the key problem that exists is, the object of these technology implementations is not wine product itself, but the external things of wine product, for the wine product, only be fittings or parasite, there is no directly with the wine product, intrinsic, necessary relation.The effect that it can play only is to have made a cover armor and a helmet for the wine product, adopt the method for stealing the beams and changing the pillars when running into deceitful dangerous counterfeiter, when fake wine is really packed, this only is that anti-dummy unit is attached on the false proof theme, false proof strategy in heavily table does not weigh under the thinking, just will inevitably stay opportunity, finally cause counterfeit and shoddy goods to confuse market order to the counterfeiter.Intension anti-counterfeiting technology at wine product itself mainly is to utilize infrared spectra, gas-chromatography and mass spectrum detection to set up the finger printing of various famous-brand and high-quality liquor, because the difference of the different residing region of liquor manufacturer, weather, microbial environment, water quality environment difference and production technique, the composition of the liquor of producing is different, carries out the evaluation of fingerprint with infrared spectra, gas-chromatography and mass spectrum according to the different mining of these compositions.This intension anti-counterfeiting technology need be set up the standard finger-print of various liquor in advance, and this just needs the strict control quality of production, guarantees that the difference between the different batches narrows down to minimum, and main chemical does not change.The DNA anti-counterfeiting technology can in time detect the advantage of identifying but can't duplicating and decode with the characteristics and the utilization proprietary technology of the complicated coding of its natural material, more and more is subjected to the favor of false proof industry, is used as the sharp keen modern weapons that hits counterfeit and shoddy goods.The DNA anti-counterfeiting technology can also can be carried out false proof at product " self " at the packing of product; when it carries out existing equally when false proof the problem of the true packing of fake wine at the packing of product; therefore the present invention adopts and add DNA in liquor; detect the DNA that adds in the liquor then; foundation is at the intension anti-counterfeiting technology of liquor " self "; this intension anti-counterfeiting technology is professional anti-counterfeiting technology; can be each liquor manufacturer; quality surveillances at different levels and market surpervision department provide technical support; allow their in right-safeguarding law enforcement initiatively; rapidly; take action protection business and consumer's legitimate rights and interests accurately.
Three, summary of the invention
Technical problem
The objective of the invention is to provides a kind of method of differentiating falsification of distilled spirit at the problem that exists in the present liquor anti-counterfeiting technology.
Technical scheme
The invention provides a kind of method of utilizing nucleic acid detection technique to differentiate falsification of distilled spirit.
The above-mentioned method of utilizing nucleic acid detection technique to differentiate falsification of distilled spirit is:
(1) in liquor, adds a kind of dna solution.
(2) adopt the method for ethanol sedimentation to extract the DNA that is added in the wine.
(3) with the DNA that extracts as template, carry out the PCR reaction with corresponding primer, get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.
(4) real and fake discrimination standard: electrophoresis result purpose band and size occur and conforms to the dna segment design load that is added and to be true wine, purpose band or size do not occur and does not conform to and be fake wine.
Beneficial effect
The invention provides a kind of method of utilizing nucleic acid detection technique to differentiate falsification of distilled spirit.For each liquor manufacturer, quality surveillances at different levels city and a supervision department provide technical support, allow they in the right-safeguarding law enforcement initiatively, rapidly, take action protection enterprise and human consumer's legitimate rights and interests accurately.
Four, description of drawings
Fig. 1 is various number of degrees liquor samples that add bacillus pumilus CGMCC No.2337 genomic dna and the pcr amplification product electrophoresis detection result who does not add the various number of degrees liquor samples of bacillus pumilus CGMCC No.2337 genomic dna.M is dna molecular amount standard (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); 1 for adding 38 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 2 for not adding 38 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 3 for adding 45 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 4 for not adding 45 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 5 for adding 52 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 6 for not adding 52 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 7 for adding 55 ° of liquor of bacillus pumilus CGMCCNo.2337 genomic dna; 8 for not adding 55 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 9 for adding 65 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 10 for not adding 65 ° of liquor of bacillus pumilus CGMCC No.2337 genomic dna; 11 is bacillus pumilus CGMCC No.2337 genome DNA sample.
Five, embodiment
Embodiment one: to add the chicken genomic dna in liquor is that example is differentiated falsification of distilled spirit
1 illustrate genomic dna in the extraction chicken according to blood/cell/tissue genome DNA extracting reagent kit (day root biochemical technology company limited).
2 add 1mg chicken genomic dna solution in 500mL liquor.
3 extract chicken genomic dna in the liquor.
(1) gets the liquor 400 μ L that contain the chicken genomic dna and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims the chicken genomic dna.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) chicken genomic dna precipitation is dissolved in the TE solution of 10 μ L pH 8.0, fully the rinsing tube wall.
4 is template with the chicken genomic dna that extracts from wine, carries out the PCR reaction with SEQ ID NO.1 in the sequence table and SEQ ID NO.2 primer, and amplification purpose band is 261bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
5 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 261bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Embodiment two: to add soybean gene group DNA in liquor is that example is differentiated falsification of distilled spirit
1 illustrate soybean gene group DNA in the fresh leaflet tablet of extraction soybean according to novel plant genome DNA extracting reagent kit (day root biochemical technology company limited).
2 add 1mg soybean gene group dna solution in 500mL liquor.
3 extract soybean gene group DNA in the liquor.
(1) gets the liquor 400 μ L that contain soybean gene group DNA and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims soybean gene group DNA.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) soybean gene group DNA precipitation is dissolved in the TE solution of 10 μ LpH 8.0, fully the rinsing tube wall.
4 is template with the soybean gene group DNA that extracts from wine, and SEQ ID NO.3 and SEQ ID NO.4 primer carry out the PCR reaction in the sequence table, and amplification purpose band is 436bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
5 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 436bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Embodiment three: to add pastoris genomic dna in liquor is that example is differentiated falsification of distilled spirit
1 extracts test kit (day root biochemical technology company limited) according to pastoris genomic dna illustrates and extracts pastoris genomic dna.
2 add 10 μ g pastoris genomic dna solution in 500mL liquor.
3 extract pastoris genomic dna in the liquor.
(1) gets the liquor 400 μ L that contain pastoris genomic dna and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims pastoris genomic dna.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) the pastoris genomic dna precipitation is dissolved in the TE solution of 10 μ L pH 8.0, fully the rinsing tube wall.
4 is template with the pastoris genomic dna that extracts from wine, carries out the PCR reaction with SEQ ID NO.5 in the sequence table and SEQ IDNO.6 primer, and amplification purpose band is 730bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
5 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 730bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Embodiment four: to add bacillus pumilus CGMCC No.2337 genomic dna in liquor is that example is differentiated falsification of distilled spirit
1 illustrate extraction bacillus pumilus CGMCC No.2337 genomic dna according to bacterial genomes DNA extraction test kit (day root biochemical technology company limited).
2 add 1 μ g bacillus pumilus CGMCC No.2337 genomic dna solution in 500mL liquor.
3 extract bacillus pumilus CGMCC No.2337 genomic dna in the liquor.
(1) gets the liquor 400 μ L that contain bacillus pumilus CGMCC No.2337 genomic dna and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims bacillus pumilus CGMCC No.2337 genomic dna.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) bacillus pumilus CGMCC No.2337 genomic dna precipitation is dissolved in the TE solution of 10 μ L pH 8.0, fully the rinsing tube wall.
4 is template with the bacillus pumilus CGMCC No.2337 genomic dna that extracts from wine, carries out the PCR reaction with SEQ ID NO.7 in the sequence table and SEQ ID NO.8 primer, and amplification purpose band is 1169bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
5 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 1169bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Embodiment five: to add the canine parvovirus genomic dna in liquor is that example is differentiated falsification of distilled spirit
1 extracts test kit (day root biochemical technology company limited) according to virus genom DNA/RNA illustrates and extracts the canine parvovirus genomic dna.
2 add 1 μ g canine parvovirus genomic dna solution in 500mL liquor.
3 extract canine parvovirus genomic dna in the liquor.
(1) gets the liquor 400 μ L that contain the canine parvovirus genomic dna and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims the canine parvovirus genomic dna.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) canine parvovirus genomic dna precipitation is dissolved in the TE solution of 10 μ L pH 8.0, fully the rinsing tube wall.
4 is template with the canine parvovirus genomic dna that extracts from wine, carries out the PCR reaction with SEQ ID NO.9 and SEQ ID NO.10, and amplification purpose band is 448bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
5 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 448bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Embodiment six: to add the pUC57 plasmid DNA in liquor is that example is differentiated falsification of distilled spirit
1 adds 1ng pUC57 plasmid DNA (Fermentas International Inc.) solution in 500mL liquor.
2 extract pUC57 plasmid DNA in the liquor.
(1) gets the liquor 400 μ L that contain the pUC57 plasmid DNA and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims the pUC57 plasmid DNA.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) pUC57 plasmid DNA precipitation is dissolved in the TE solution of 10 μ LpH 8.0, fully the rinsing tube wall.
3 is template with the pUC57 plasmid DNA of extracting from wine, carries out the PCR reaction with SEQ ID NO.11 and SEQ ID NO.12, and amplification purpose band is 174bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
4 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 174bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Embodiment seven: to add the pig genomic DNA fragment in liquor is that example is differentiated falsification of distilled spirit
1 illustrate genomic dna in the extraction pork according to blood/cell/tissue genome DNA extracting reagent kit (day root biochemical technology company limited).
2 genomic dnas with the pig of extracting are template, carry out the PCR reaction with SEQ ID NO.13 and SEQ ID NO.14, and amplification purpose band is 600bp.25 μ LPCR reaction systems are: 2 * PCRMix12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 1 μ L, moisturizing to 25 μ L.Loop parameter is: 94 ℃, and 2 pre-sex change min.94 ℃ of sex change 15s, 55 ℃ of annealing 30s, 68 ℃ are extended 1min, repeat 10 circulations; 94 ℃ of sex change 15s, 50 ℃ of annealing 30s, 68 ℃ are extended 1min, repeat 10 circulations; 94 ℃ of sex change 15s, 45 ℃ of annealing 30s, 68 ℃ are extended 1min, repeat 10 circulations; 68 ℃ are extended 5min.
3 illustrates recovery PCR product according to common DNA product purification test kit (day root biochemical technology company limited).
4 add 10ng PCR product in 500mL liquor.
5 extract PCR product in the liquor.
(1) gets the liquor 400 μ L that contain the PCR product and place the 1.5mL centrifuge tube, add the sodium acetate soln 40 μ L of 3.0mol/L, fully mixing.
(2) add the ice-cold dehydrated alcohol of 900 μ L, add the magnesium chloride solution 14 μ L of 1mol/L again, fully mixing.Then as for 1h on ice.
(3) 0 ℃, 12000r/min, centrifugal 20min reclaims the PCR product.
(4) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(5) add 70% the ethanol of 700 μ L, in 4 ℃, 12000r/min, centrifugal 20min.
(6) carefully shift out supernatant liquor with automatic micropipettor, exhaust all drops that invest tube wall.
(7) centrifuge tube is placed on the experiment table in the room temperature lower open mouth, volatilizes until residual liquid.
(8) PCR product precipitation is dissolved in the TE solution of 10 μ L pH 8.0, fully the rinsing tube wall.
6 is template with the PCR product that extracts from wine, carries out the PCR reaction with SEQ ID NO.15 and SEQ ID NO.16, and amplification purpose band is 364bp.
25 μ LPCR reaction systems are: 2 * PCRMix (day root biochemical technology company limited), 12.5 μ L, each 1 μ L of 10 μ mol/L upstream and downstream primers, template DNA 5 μ L, moisturizing to 25 μ L.
Loop parameter is: 94 ℃, and pre-sex change 5min.94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 10 circulations, and each cycle annealing temperature is fallen 0.5 ℃; 94 ℃ of sex change 15s then, 50 ℃ of annealing 30s, 72 ℃ are extended 1min, repeat 20 circulations; Last 72 ℃ are extended 5min.
7 get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, and 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.The purpose band that 364bp appears in electrophoresis result is true wine, purpose band or size do not occur and does not conform to and be fake wine.
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉a kind of method of utilizing nucleic acid detection technique to differentiate falsification of distilled spirit
＜130〉specification sheets
<160>16
<170>PatentIn?version?3.1
<210>1
<211>22
<212>DNA
＜213〉synthetic
<400>1
tggacttaac?actgctattg?cc 22
<210>2
<211>21
<212>DNA
＜213〉synthetic
<400>2
ctctctacct?tggagggctg?a 21
<210>3
<211>18
<212>DNA
＜213〉synthetic
<400>3
cttcgccgct?tccttcaa 18
<210>4
<211>19
<212>DNA
＜213〉synthetic
<400>4
gagtcccgtg?gcagcagag 19
<210>5
<211>28
<212>DNA
＜213〉synthetic
<400>5
gggatgtatt?tattagataa?aaaatcaa 28
<210>6
<211>24
<212>DNA
＜213〉synthetic
<400>6
gcagtagtta?gtcttcagta?aatc 24
<210>7
<211>30
<212>DNA
＜213〉synthetic
<400>7
ccgtctagaa?tgtgcgtaaa?aaagaaaaat 30
<210>8
<211>26
<212>DNA
＜213〉synthetic
<400>8
tcggatcctt?agttagaagc?tgcttg 26
<210>9
<211>18
<212>DNA
＜213〉synthetic
<400>9
cacaagcggc?aagcaatc 18
<210>10
<211>18
<212>DNA
＜213〉synthetic
<400>10
gcggcgtcag?aagggtta 18
<210>11
<211>24
<212>DNA
＜213〉synthetic
<400>11
cgccagggtt?ttcccagtca?cgac 24
<210>12
<211>24
<212>DNA
＜213〉synthetic
<400>12
gagcggataa?caatttcaca?cagg 24
<210>13
<211>25
<212>DNA
＜213〉synthetic
<400>13
tgtcttcttc?ctcagtggtc?gtgct 25
<210>14
<211>24
<212>DNA
＜213〉synthetic
<400>14
ccaatcagca?ggctcatggt?acaa 24
<210>15
<211>21
<212>DNA
＜213〉synthetic
<400>15
ggctgcactg?cttgaaggca?c 21
<210>16
<211>20
<212>DNA
＜213〉synthetic
<400>16
ggctctgggc?tccatgaggg 20
Claims (3)
1. method of utilizing nucleic acid detection technique to differentiate falsification of distilled spirit is characterized in that:
(1) in liquor, adds a kind of dna solution.
(2) adopt the method for ethanol sedimentation to extract the DNA that is added in the wine.
(3) with the DNA that extracts as template, carry out the PCR reaction with corresponding primer, get PCR product 7 μ L, adopt the sepharose of mass ratio 2.0% to contain 0.005%GOLDView, 0.5 * TBE electrophoretic buffer, 120V constant voltage electrophoresis 40min, ultraviolet lamp observe electrophoresis result down.
(4) real and fake discrimination standard: electrophoresis result purpose band and size occur and conforms to the dna segment design load that is added and to be true wine, purpose band or size do not occur and does not conform to and be fake wine.
2. the method for discriminating falsification of distilled spirit as claimed in claim 1 is characterized in that: described DNA is from the genomic dna or the gene fragment of animal, plant, microorganism or synthetic.
3. the application of the method for discriminating falsification of distilled spirit as claimed in claim 1 aspect all brand falsification of distilled spirit discriminatings.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910035889A CN101665825A (en) | 2009-10-09 | 2009-10-09 | Method for identifying falsification of distilled spirit by utilizing nucleic acid detection technique |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910035889A CN101665825A (en) | 2009-10-09 | 2009-10-09 | Method for identifying falsification of distilled spirit by utilizing nucleic acid detection technique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101665825A true CN101665825A (en) | 2010-03-10 |
Family
ID=41802600
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910035889A Pending CN101665825A (en) | 2009-10-09 | 2009-10-09 | Method for identifying falsification of distilled spirit by utilizing nucleic acid detection technique |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101665825A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102384947A (en) * | 2011-08-03 | 2012-03-21 | 中国食品发酵工业研究院 | Method for distinguishing genuineness of solid-fermentation liquor |
| CN106498072A (en) * | 2016-11-24 | 2017-03-15 | 青岛千卓分子生物科技有限公司 | The guard method and its application of foreign DNA internal standard compound in a kind of liquid form product |
| CN109628565A (en) * | 2018-12-28 | 2019-04-16 | 江苏权正检验检测有限公司 | A method of building genome is to identify that white wine is true and false |
| WO2020109597A1 (en) | 2018-11-30 | 2020-06-04 | Orvinum Ag | Method for providing an identifier for a product |
-
2009
- 2009-10-09 CN CN200910035889A patent/CN101665825A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102384947A (en) * | 2011-08-03 | 2012-03-21 | 中国食品发酵工业研究院 | Method for distinguishing genuineness of solid-fermentation liquor |
| CN102384947B (en) * | 2011-08-03 | 2014-06-11 | 中国食品发酵工业研究院 | Method for distinguishing genuineness of solid-fermentation liquor |
| CN106498072A (en) * | 2016-11-24 | 2017-03-15 | 青岛千卓分子生物科技有限公司 | The guard method and its application of foreign DNA internal standard compound in a kind of liquid form product |
| CN106498072B (en) * | 2016-11-24 | 2019-09-10 | 青岛千卓分子生物科技有限公司 | The guard method and its application of exogenous DNA internal standard compound in a kind of liquid form product |
| WO2020109597A1 (en) | 2018-11-30 | 2020-06-04 | Orvinum Ag | Method for providing an identifier for a product |
| CN109628565A (en) * | 2018-12-28 | 2019-04-16 | 江苏权正检验检测有限公司 | A method of building genome is to identify that white wine is true and false |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101712996A (en) | Method for quickly identifying categories of meat and dried meat products of five domestic animals | |
| CN101665825A (en) | Method for identifying falsification of distilled spirit by utilizing nucleic acid detection technique | |
| CN101845495A (en) | Gene chip of inspection and quarantine fruit fly | |
| Tai et al. | Temporal and spatial distributions of marine Synechococcus in the Southern California Bight assessed by hybridization to bead-arrays | |
| Varga et al. | Characterization of Pasteurella multocida strains isolated from geese | |
| Martinez-Fernandez et al. | Sample processing methods impacts on rumen microbiome | |
| CN101962677A (en) | Method for identifying poultry gender | |
| CN102242220B (en) | Polymerase chain reaction (PCR) primer pair for identifying or assisting in identifying duck tissues and/or organs and application of PCR primer pair | |
| CN101177714A (en) | Monocyte hyperplasia Listeria PCR detecting method adding amplified interior label | |
| CN105177151B (en) | A kind of DNA bar code primer pair, kit and method for differentiating Chinese yew kind | |
| CN102337347A (en) | Characteristic nucleotide sequence for identifying ophiocordyceps crinalis, as well as probes and method thereof | |
| CN103614484A (en) | Specific PCR (Polymerase Chain Reaction) identification method of paecilomyces hepiali powder | |
| CN111057775A (en) | Specific novel molecular target for identifying salmonella and rapid detection method thereof | |
| CN103436622B (en) | Method as well as molecular marker and primer for identifying rice male sterility type and three-line hybrid rice type | |
| CN102312011A (en) | PCR primer pair for identification or auxiliary identification of tissues and/or organs of mouse and applications thereof | |
| CN101805800B (en) | Method for distinguishing indigenous pig breed in China from indigenous pig breed in Europe | |
| CN101109019A (en) | Method for detecting vibrio parahaemolyticus PCR added with amplification internal sign | |
| CN105331677A (en) | Primer group used for multiple PCR detection by aiming at salmonella and Shigella in food, kit and detection method thereof | |
| CN106319080A (en) | PCR detection kit for rapidly identifying mycoplasma hyopeumoniae, mycoplasma synoviae and mycoplasma hyorhinis, and applications of PCR detection kit | |
| CN1275717A (en) | Nucleic acid molecular probe for identifying blue-green alga and method | |
| CN103602754A (en) | Specific PCR (Polymerase Chain Reaction) identification method of cordyceps militaris | |
| CN104774938A (en) | Identification method and identification kit of six cyprinid fishes and raw meat products on basis of DNA (deoxyribonucleic acid) Barcoding | |
| CN1944665B (en) | Primer sequence and detecting reagent kit for detecting delayed Edward's bacteria | |
| CN110923349A (en) | Species-specific detection molecular tags 3283 and 3316 of yersinia enterocolitica and rapid detection method thereof | |
| CN105331675B (en) | For multiplex PCR detection primer sets, kit and the detection method of the staphylococcus aureus in food |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20100310 |