CN104561328B - The kit of dog derived component and its application in a kind of detection food - Google Patents
The kit of dog derived component and its application in a kind of detection food Download PDFInfo
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- CN104561328B CN104561328B CN201510024486.7A CN201510024486A CN104561328B CN 104561328 B CN104561328 B CN 104561328B CN 201510024486 A CN201510024486 A CN 201510024486A CN 104561328 B CN104561328 B CN 104561328B
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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
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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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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention provides a kind of kit for detecting dog 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 dog 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 dog 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 dog derived component in food kit 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.
Dog meats have received very big concern in recent years as the carnivorous source of a kind of tradition in Chinese dietetic, some pet dogs
It is slaughtered to be mixed into meat products, cause the extensive concern of society.Therefore, work out it is a kind of accurate, quickly and reliably differentiate one
The method for planting fast and accurately detection dog 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 dog derived component is there is not yet report.Therefore, set up
Dog derived component real-time fluorescence PCR detection method in animal derived product, to improving accident Ability of emergency management, lifting food
Product security risk level monitoring and ability to supervise are significant.
The content of the invention
It is an object of the invention to provide a kind of kit for detecting dog derived component in food and its application.
Present invention firstly provides primer pair and fluorescence probe for detecting dog derived component in food, its nucleotides sequence
Row are respectively as shown in SEQ ID NO.1-3.
The invention provides the application of above-mentioned primer pair and fluorescence probe in dog derived component in detecting food.
The invention provides above-mentioned primer pair and fluorescence probe in dog derived component kit in preparing detection food
Using.
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 dog 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.
The invention provides a kind of method for detecting dog derived component in food, the method is extracted with RNA extraction based on proteinase K digestion
Food in DNA, with it as template, using the present invention provide primer pair and fluorescence probe carry out fluorescent quantitative PCR, root
According to Ct value result of determination.
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 dog source in food
Property composition detection provide new method.Fluorescence RCR technologies of the present invention can be realized quickly and accurately in food
The discriminating detection of dog derived component, can complete in 1h~2h, have the advantages that quick, special, sensitive, high flux, minimum inspection
Survey limit and be about 100fg, the ability with derived component in good detection food can effectively resist that meat products is adulterated, and it is right to increase
The protection of consumer's interests.
Brief description of the drawings
Fig. 1 is the specificity verification result of the fluorescence quantifying PCR method of dog derived component in present invention detection food, application
The method of the present invention only has positive amplification to dog dna, and the DNA detections of other 24 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 a large amount of genes of ND 2 for comparing dog in GenBank, the selection genes of ND 2 are highly conserved and with species specificity
Gene order is template, designs the specific upstream and downstream primers of dog ND 2 and Taqman probes, and upstream and downstream primer length is respectively
The nucleotides of 19bp and 25bp, probe length is the nucleotides of 28bp, and in 5 ' the upper FAM fluorophors of end mark, 3 ' end marks
Upper TAMRA quenching groups, sequence is as follows：
Sense primer：5`-CTCCGGCCAATGGGTAATC-3`(SEQ ID NO.1)
Anti-sense primer：5`-GAAGTGGAATGGAGATAGGCCTAGT-3`(SEQ ID NO.2)
Fluorescence probe：5`-FAM-TCAAACCCCATCGCATCCATCATGATAA-TAMRA-3`(SEQ ID NO.3)
The foundation of the fluorescent quantitative PCR detection method of embodiment 2
1st, sample DNA is extracted
(1) dog meats sample 0.2g 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, the dog dna that said extracted is obtained is template, is sun with dog genomic DNA
Property control, with free nucleic acid distilled water as negative control, set up fluorescence quantitative PCR detection system.
Its 20 μ L total working system is：
Its working procedure is：95 DEG C of predegeneration 10min, 1 circulation；95 DEG C of denaturation 15sec, 60 DEG C of anneal 1min, 40
Circulation.
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 >=35.0 is feminine gender.
The specific test of embodiment 3
To verify the specificity of this kit, with domesticated dog genomic DNA as positive control, with pig, ox, sheep, goat,
Chicken, duck, pigeon, turkey, quail, ostrich, grey goose, cat, mouse, roe deer, horse, donkey, fox, camel, salmon, perch, crucian,
Sika deer, rainbow trout, grass carp.Totally 24 species are negative control, with dd H2O is blank, sets up glimmering using embodiment 2
Fluorescent Quantitative PCR detection architecture is detected to above-mentioned 25 species.After amplification terminates, same threshold is taken after deducting background fluorescence signal
Value analyze data, determines the Ct values of each sample.Experimental result is shown in Fig. 1, table 1, and the Ct values of dog are 11.19, are shown as positive findings,
The CT values of remaining 24 species are all higher than 35, are shown as negative findings.Illustrate that the experiment proves that this kit has good thing
Species specificity.
The inventive method of table 1 is to 25 kinds of testing results of animal DNA
The sensitivity test of embodiment 4
To verify the sensitivity of this kit, it is by domesticated dog genomic DNA gradient dilution：1ng/ μ l, 100pg/ μ l,
The dilution of 10pg/ μ l, 1pg/ μ l, 100fg/ μ l, 10fg/ μ l, and using these dilutions as positive control, it is double with free nucleic acid
Steaming water is negative control, and 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 1.Result shows
The dog derived component of the detectable about 100fg of the fluorescence quantifying PCR method of dog derived component in detection food of the invention.Explanation
The inventive method has good sensitivity.
Table 2
| DNA content | 1ng | 100pg | 10pg | 1pg | 100fg | 10fg |
| Ct values | 18.79 | 22.09 | 25.49 | 28.82 | 32.51 | 35.51 |
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 (6)
1. it is a kind of to detect dog derived component kit in food, it is characterised in that to contain nucleotide sequence such as SEQ ID NO.1-2
Fluorescence probe of the shown primer pair and nucleotide sequence as shown in SEQ ID NO.3.
2. kit as claimed in claim 1, 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 dog genomic DNA.
3. kit as claimed in claim 1, it is characterised in that its 20 μ L PCR reaction system is：
4. kit as claimed in claim 1, it is characterised in that its working procedure is：95 DEG C of predegeneration 10min；95 DEG C of changes
Property 15s, 60 DEG C annealing 1min, 40 circulation.
5. it is a kind of detect food in dog derived component method, it is characterised in that the food that the method is extracted with RNA extraction based on proteinase K digestion
DNA in product, with it as template, primer pair and nucleotide sequence such as SEQ using nucleotide sequence as shown in SEQ ID NO.1-2
Fluorescence probe shown in ID NO.3 carries out fluorescent quantitative PCR, according to Ct value result of determination.
6. the primer pair and nucleotide sequence containing nucleotide sequence as shown in SEQ ID NO.1-2 are as shown in SEQ ID NO.3
Fluorescence probe diagnostic reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510024486.7A CN104561328B (en) | 2015-01-16 | 2015-01-16 | The kit of dog derived component and its application in a kind of detection food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510024486.7A CN104561328B (en) | 2015-01-16 | 2015-01-16 | The kit of dog derived component and its application in a kind of detection food |
Publications (2)
| Publication Number | Publication Date |
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| CN104561328A CN104561328A (en) | 2015-04-29 |
| CN104561328B true CN104561328B (en) | 2017-07-04 |
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| CN201510024486.7A Expired - Fee Related CN104561328B (en) | 2015-01-16 | 2015-01-16 | The kit of dog derived component and its application in a kind of detection food |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104988233B (en) * | 2015-07-14 | 2018-01-30 | 山东省农业科学院畜牧兽医研究所 | A kind of LAMP detection method for differentiating dog meats in beef and mutton using mitochondrial DNA |
| CN107488708A (en) * | 2016-06-12 | 2017-12-19 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for dog derived component digital pcr |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775436A (en) * | 2009-12-23 | 2010-07-14 | 吉林大学 | Method for detecting bovine and sheep derived materials in feed and kit |
| CN103923979A (en) * | 2014-03-19 | 2014-07-16 | 毕节学院 | PCR detection primer kit for identification detection of dog source component, and detection method thereof |
-
2015
- 2015-01-16 CN CN201510024486.7A patent/CN104561328B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775436A (en) * | 2009-12-23 | 2010-07-14 | 吉林大学 | Method for detecting bovine and sheep derived materials in feed and kit |
| CN103923979A (en) * | 2014-03-19 | 2014-07-16 | 毕节学院 | PCR detection primer kit for identification detection of dog source component, and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| AB499825.1;GenBank;《GenBank》;20141009 * |
| GB/T 21105-2007 动物源性饲料中狗源性成分定性检测方法 实时荧光PCR方法;中华人民共和国国家质量监督检验检疫总局、中国国家标准化管理委员会;《中华人民共和国国家标准》;20071024;第1-3页 * |
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