CN104561329B - The kit of roe deer derived component and its application in a kind of detection food - Google Patents
The kit of roe deer derived component and its application in a kind of detection food Download PDFInfo
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- CN104561329B CN104561329B CN201510024602.5A CN201510024602A CN104561329B CN 104561329 B CN104561329 B CN 104561329B CN 201510024602 A CN201510024602 A CN 201510024602A CN 104561329 B CN104561329 B CN 104561329B
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- Prior art keywords
- food
- roe deer
- derived component
- primer pair
- detection
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Classifications
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
Abstract
The invention provides a kind of kit for detecting roe deer derived component in food and its application, belong to technical field of food detection.Whether kit of the invention carries out fluorescence quantitative PCR detection using the fluorescence probe shown in the primer shown in SEQ ID NO.1 2 and SEQ ID NO.3 to food to be measured, judges contain roe deer derived component in food according to Ct values.Kit of the present invention has detection accurate, and sensitivity is high, high specificity, simple and rapid advantage; lowest detection is limited to 100fg; ability with roe deer derived component in good detection food, can effectively resist that meat products is adulterated, increase the protection to consumer's interests.
Description
Technical field
The present invention relates to technical field of food detection, relate in particular to detect the kit of roe deer derived component in food
And its application.
Background technology
Ratio of the animal derived food in people's ordinary meal is incrementally increased, various cold fresh meats, intensive processing half into
The consumption proportions such as meat, cooked meat product are savored to rise year by year.But be present very big difference in the meat and price of different plant species, mixed to doping
Vacation creates great profit margin to seek economic interests, and some illegal enterprises and retailer use low cost in meat products
Meat happens occasionally instead of the phenomenon of high price meat.The health and rights and interests of this not only serious infringement consumer, can also be related to religion
Faith, cause ethnic minority issue, while directly influence country image, society harmonious development and government public credibility.
Roe deer has abundant nutrition, and delicate delicious mouthfeel as a kind of carnivorous source, in China particularly north
Just there are vast consumer groups in side area.But the roe deer of in the market is very different, true and false difficult point, therefore, work out a kind of standard
Really, the method for quickly and reliably differentiating roe deer derived component, is just highly desirable to.
As application of the molecular biology method in Food Inspection deepens continuously, the discriminating of animal derived materials in food
Technology is also evolving, and differentiates the continuous improvement of precision and detection sensitivity, has preliminarily formed at present and is with genetic test
The method system on basis.Technology based on PCR (PCR) has turned into animal derived materials identification in food
Core methed.Species specificity PCR designs specific primer according to the difference site of different plant species gene order, using PCR
The exponential amplification of animal derived materials characterizing gene fragment is realized in reaction, then by electrophoresis detection differentiate possible species into
Point.Multiple species are detected simultaneously during multiplex PCR can realize mixture.In recent years, real-time fluorescence PCR technology be it is animal derived into
The method of most study in go-on-go survey, compared with regular-PCR, with high specificity, sensitivity high, effectively favorable reproducibility, reduction
The advantages of pollution.
Mammalian mitochondria DNA (mtDNA) is relatively independent in heredity, with genome it is small, without repetitive sequence, life
The features such as in the internal inorganization specificity of thing, each cell containing a large amount of mitochondrial genomes.Therefore, with mtDNA molecule marks
Note differentiates animal derived materials compared with core DNA molecular marker, with sensitivity is high, the advantage such as accuracy is good, DNA degradation is small.
Fluorescent PCR detection technique based on animal mitochondria DNA has broad application prospects in animal derived materials discriminating.
At present, Species-specific primer is designed according to mitochondrial genomes DNA sequence dna difference, sets up PCR and real-time fluorescence
PCR method has been shown in a large amount of reports, however, the fluorescence PCR method for the detection of roe deer derived component is there is not yet report.Therefore, build
Roe deer derived component real-time fluorescence PCR detection method in animal derived product is found, to improving accident Ability of emergency management, is carried
Rise food safety risk supervision level and ability to supervise is significant.
The content of the invention
It is an object of the invention to provide a kind of kit for detecting roe deer derived component in food and its application.
Present invention firstly provides primer pair and fluorescence probe for detecting roe deer derived component in food, its nucleotides
Sequence is respectively as shown in SEQ ID NO.1-3.
The invention provides the application of above-mentioned primer pair and fluorescence probe in roe deer derived component in detecting food.
The invention provides above-mentioned primer pair and fluorescence probe in roe deer derived component kit in preparing detection food
Application.
Kit containing primer pair of the present invention and fluorescence probe falls within protection scope of the present invention.Reagent of the invention
Box can be formed by multiple cut-off, and one or more are fixed such as pipe or the container of bottle to accommodate.One of these containers or
Person's multiple can be equipped with primer of the invention and fluorescence probe, and the primer and fluorescence probe can be lyophilized forms as needed
Or it is dissolved in the state in buffer solution.In addition, the one kind for quantitative fluorescent PCR reaction can also be included in kit of the invention
Or other compositions and apparatus required for various enzyme/reagents, and the implementation present invention, such as DNA lysates, fluorescent quantitation reaction
Liquid, negative template, positive template, the negative template are distilled water, and the positive template is roe deer genomic DNA.
Further, its 20 μ L work system of kit of the invention is：
The working procedure of kit of the present invention is：95 DEG C of 10min of predegeneration；Again through 95 DEG C of denaturation 15s, 60 DEG C of annealing
1min, 40 circulations.
Negative control and positive control must be set up during the sample of detection every time of the invention, two kinds of controls in the detection are effectively expansion
During increasing, sample results criterion is as follows：
Ct values<Sample result is the positive when 35.0；
The sample result of Ct value >=35.0 is feminine gender.
A kind of method of roe deer derived component in detection food, in the food that the method is extracted with RNA extraction based on proteinase K digestion
DNA, with it as template, the primer pair and fluorescence probe provided using the present invention carry out fluorescent quantitative PCR, are sentenced according to Ct values
Determine result.
Diagnostic reagent containing primer pair of the present invention and probe falls within protection scope of the present invention.
Kit specificity of the present invention is good, and detection method is quick and easy, and accuracy is high, and sensitivity is good, is roe deer in food
The detection of derived component provides new method.Fluorescence RCR technologies of the present invention can be realized quickly and accurately to food
The discriminating detection of middle roe deer derived component, can complete in 1h~2h, have the advantages that quick, special, sensitive, high flux, no
It is only capable of providing new method for detection roe deer derived food, and can effectively resists that meat products is adulterated, increasing is to consumer's interests
Protection.
Brief description of the drawings
Fig. 1 is the specificity verification result of the fluorescence quantifying PCR method of roe deer derived component in present invention detection food, should
Only there is positive amplification to roe deer DNA with the method for the present invention, the DNA detections of other 25 species are feminine gender.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modification or replacement made to the inventive method, step or condition belong to the scope of the present invention.
If not specializing, chemical reagent used is conventional commercial reagent in embodiment, skill used in embodiment
The conventional meanses that art means are well known to those skilled in the art.
The design of the primer of embodiment 1
Through CYTB genes in a large amount of comparison GenBank, selection CYTB genes are highly conserved and with species-specific gene
Sequence is template, designs roe deer CYTB specific primers pair and Taqman probes, is respectively designated as roe deer CYTB-F (P1), roe deer
CYTB-R (P2), roe deer CYTB-Probe (Probe-P3), for the primer and probe sequence of Taqman real-time fluorescent PCR amplifications
It is as follows：
Roe deer CYTB-F：5`-AGCAATACATTACACATCCGACACA-3`(SEQ ID NO.1)
Roe deer CYTB-R：5`-AAATATTGATGCTCCGTTTGCA-3`(SEQ ID NO.2)
Roe deer CYTB-P3：5`-FAM-AGCATTCTCCTCTGTTACTCACATCTGCCG-TAMRA-3`(SEQ ID
NO.3)
The foundation of the fluorescent quantitative PCR detection method of embodiment 2
1st, sample DNA is extracted
(1) roe deer this 0.2g of meat sample is taken, is shredded as far as possible.It is placed in 1.5ml centrifuge tubes and adds the cell lysis buffer solution of 1ml,
20 μ l Proteinase Ks (500 μ g/ml), mix.The water-bath 30min in 65 DEG C of thermostat water baths, intermittent oscillation centrifuge tube is for several times.In
Desk centrifuge is centrifuged 5min with 12,000rpm, takes during supernatant enters another centrifuge tube.
(2) isometric phenol is added:Chloroform mixed liquor (1:1), vibration is mixed, 12,000rpm centrifugation 10min.
(3) supernatant to another pipe is taken, isometric chloroform is added, vibration is mixed, 12,000rpm centrifugation 10min.
(4) supernatant to another pipe is taken, the 3mol/L sodium acetates and 2 times of absolute ethyl alcohols of volume of 1/10 volume is added, is mixed
Even rear precipitation at room temperature 10min, 12,000rpm centrifugation 10min.
(5) supernatant is abandoned, precipitation, 12,000rpm centrifugation 5min is washed with the ethanol of 1ml 75%.
(6) repeat step 5.
(7) supernatant is abandoned, drying at room temperature 5min will be precipitated to.
(8) 50 μ l TE or dd H are added2O dissolution precipitations, are subsequently placed in 4 DEG C or -20 DEG C save backup.
2nd, using the primer and probe of embodiment 1, above-mentioned DNA is template, and genetic engineering side is passed through with roe deer CYTB genes
Plasmid standard obtained in method is positive control, with free nucleic acid distilled water as negative control, sets up fluorescence quantitative PCR detection body
System.
Its 20 μ L total working system is：
Its working procedure is：95 DEG C of 10min of predegeneration；Again through 95 DEG C of denaturation 15s, 60 DEG C of annealing 1min, 40 circulations
After amplification terminates, same Threshold Analysis data are taken after deducting background fluorescence signal, determine the Ct values of each sample.
Negative control and positive control must be set up during detection sample every time, when two kinds of controls are for effective amplification in the detection,
Sample results criterion is as follows：
Ct values<Sample result is the positive when 35.0；The sample result of Ct value >=350.0 is feminine gender.
The specific test of embodiment 3
To verify the specificity of this kit, with roe deer genomic DNA as positive control, with pig, ox, sheep, goat, chicken,
Duck, pigeon, quail, turkey, ostrich, grey goose, cat, mouse, domesticated dog, rabbit, horse, fox, mink, camel, deer, salmon, rainbow
Trout, perch, crucian, the genomic DNA of grass carp is detection object, and the fluorescence quantifying PCR method set up using embodiment 2 is tested
Demonstrate,prove the specificity of the inventive method.
After amplification terminates, same Threshold Analysis data are taken after deducting background fluorescence signal, determine the Ct values of each sample.Experiment
Result is shown in Fig. 1, as a result shows that the Ct values of roe deer are 16.34, and the CT values of remaining 25 species are all higher than 35, and the experiment proves this examination
Agent box has good species specificity.
The inventive method of table 1 is to 26 kinds of testing results of animal DNA
The sensitivity test of embodiment 4
To verify the sensitivity of this kit, it is by roe deer genomic DNA gradient dilution：1ng/ μ l, 100pg/ μ l, 10pg/ μ
The dilution of l, 1pg/ μ l, 100fg/ μ l, 10fg/ μ l, and using these dilutions as positive control, be with free nucleic acid distilled water
Negative control, the fluorescence quantifying PCR method set up using embodiment 2 verifies the sensitivity of the inventive method.
After amplification terminates, same Threshold Analysis data are taken after deducting background fluorescence signal, determine the Ct values of each sample.Each
Dilution gradient sets 10 parallel sampleses, and experimental result is 10 average values of sample Ct values.Experimental result is shown in Table 2.Result shows
The roe deer derived component of the detectable 100fg of the fluorescence quantifying PCR method of roe deer derived component in detection food of the invention.Say
Bright the inventive method has good sensitivity.
Table 2
| DNA content | 1ng | 100pg | 10pg | 1pg | 100fg | 10fg |
| Ct values | 18.64 | 21.98 | 25.53 | 28.67 | 32.7 | 35.58 |
Ct values are 10 average values of parallel samples Ct values in form
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. the primer pair of roe deer derived component in food is detected, it is characterised in that the nucleotide sequence of the primer pair such as SEQ
Shown in ID NO.1-2.
2. the fluorescence probe for being used cooperatively with the primer pair described in claim 1, it is characterised in that the nucleotides sequence of fluorescence probe
Row are as shown in SEQ ID NO.3.
3. the fluorescence probe described in the primer pair and claim 2 described in claim 1 detect food in roe deer derived component
In application.
4. the fluorescence probe described in the primer pair and claim 2 described in claim 1 prepare detection food in roe deer source property
Application in ingredient agent box.
5. roe deer derived component kit in a kind of detection food, it is characterised in that containing the primer pair described in claim 1 and
Fluorescence probe described in claim 2.
6. kit as claimed in claim 5, it is characterised in that also include：DNA lysates, fluorescent quantitation reaction solution, feminine gender
Template, positive template, the negative template are distilled water, and the positive template is roe deer genomic DNA.
7. it is a kind of detect food in roe deer derived component method, it is characterised in that the method is extracted with RNA extraction based on proteinase K digestion
DNA in food, with it as template, is carried out glimmering using the fluorescence probe described in the primer pair and claim 2 described in claim 1
Fluorescent Quantitative PCR is expanded, according to Ct value result of determination.
8. the detection reagent of the fluorescence probe described in the primer pair and claim 2 described in claim 1 is contained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510024602.5A CN104561329B (en) | 2015-01-16 | 2015-01-16 | The kit of roe deer derived component and its application in a kind of detection food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510024602.5A CN104561329B (en) | 2015-01-16 | 2015-01-16 | The kit of roe deer derived component and its application in a kind of detection food |
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| Publication Number | Publication Date |
|---|---|
| CN104561329A CN104561329A (en) | 2015-04-29 |
| CN104561329B true CN104561329B (en) | 2017-06-30 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201510024602.5A Expired - Fee Related CN104561329B (en) | 2015-01-16 | 2015-01-16 | The kit of roe deer derived component and its application in a kind of detection food |
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Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104862311A (en) * | 2015-05-05 | 2015-08-26 | 广州维伯鑫生物科技有限公司 | Real-time fluorescence quantification PCR primer for detecting Chinese alligator based on Taqman probe, kit and method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311998A (en) * | 2011-08-31 | 2012-01-11 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Horse or horse source component real time fluorescent PCR detection method and primers and probe for detection |
-
2015
- 2015-01-16 CN CN201510024602.5A patent/CN104561329B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102311998A (en) * | 2011-08-31 | 2012-01-11 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Horse or horse source component real time fluorescent PCR detection method and primers and probe for detection |
Non-Patent Citations (2)
| Title |
|---|
| Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction;Mohd Hazim Mohd Yusop等;《Food Anal. Methods》;20121231(第5期);第422-429页 * |
| Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene;V. Fajardo等;《Meat Science》;20071231(第76期);第235-236页 * |
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Granted publication date: 20170630 Termination date: 20190116 |