CN107858442A - Yak, the detection primer and method of three kinds of derived components of ox and buffalo in a kind of beef food - Google Patents
Yak, the detection primer and method of three kinds of derived components of ox and buffalo in a kind of beef food Download PDFInfo
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- CN107858442A CN107858442A CN201711131754.0A CN201711131754A CN107858442A CN 107858442 A CN107858442 A CN 107858442A CN 201711131754 A CN201711131754 A CN 201711131754A CN 107858442 A CN107858442 A CN 107858442A
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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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- 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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Abstract
The invention discloses yak in a kind of beef food, the detection primer and method of three kinds of derived components of ox and buffalo.The primer includes primer GTB F and primer GTB R, wherein, GTB F nucleotide sequence is as shown in SEQ ID NO.1；GTB R nucleotide sequences are as shown in SEQ ID NO.2.This method is with above-mentioned primer, carries out quantitative pcr amplification to sample to be tested, obtains QPCR amplification datas；HRM data analyses detection amplified production is carried out using quantitative PCR apparatus software kit.The present invention applies to high-resolution melting curve genotyping technique in three kinds of different calf-derived Cyclospora detections, greatly reduces the reagent consumables cost of coherent detection, while shortens detection time, and can improve the degree of accuracy of detection, avoids false positive.
Description
Technical field
The invention belongs to gene engineering technology field, specifically, is related to yak, ox and buffalo in a kind of beef food
The detection primer and method of three kinds of derived components.
Background technology
Yak, ox and buffalo belong to the different subspecies of Bos (or Bubalus), and its mitochondrial DNA similarity reaches
99%, only extremely least a portion of base has differences.Detection for calf-derived Cyclospora in beef food, prior art employ
Taqman hydrolysis probes fluorescence quantitative PCR methods, and have matched three inlet and outlet professional standards, detect respectively ox source property into
Point, buffalo derived component and yak derived component.This method is protected for yak, ox and the respective mitochondrial DNA of buffalo respectively
Keep respective a pair of the specific primers of sequences Design and Taqman probes.When detecting sample, the primer and probe of positive
Enter performing PCR to expand and there is fluorophor hydrolysis to produce fluorescence signal, the amplification curve produced by detecting fluorescence signal can be sentenced
Breaking, it is positive.And negative sample is detected by primer and probe will not be expanded institute either with or without fluorescence signal,
Fluorescence signal curve is not produced.Therefore, for each measuring samples, to carry out quantitative PCR three times and analyze its knot respectively
Fruit.
Prior art shortcoming mainly has following three points：First, cost is high, and three are detected respectively using Taqman probe techniques
Kind calf-derived Cyclospora, three pairs of different primer and probes are synthesized, and use the PCR premixed liquids of triplication and the PCR of triplication
Plate, cost are high.Second, specificity is poor, and false positive is more.The inlet and outlet professional standard formed according to prior art, it draws
All there is obvious non-specific amplification in the specificity of thing and probe, along with Taqman hydrolysis probes have no PCR in itself
Expand the defects of fluorophor that still comes off excites fluorescence signal so that testing result false positive is very serious.3rd, operation is multiple
It is miscellaneous, take.Because one sample of every detection will configure three different PCR system solution, therefore the operating time is grown.
The content of the invention
In view of this, the present invention is directed to the problem of above-mentioned, there is provided three kinds of yak, ox and buffalo in a kind of beef food
The detection primer and method of derived component, the present invention utilize the base in the conservative section of itself mitochondrial DNA of yak, ox and buffalo
Difference, discriminating detection quickly and accurately is carried out to three kinds of different calf-derived Cyclosporas of beef food.
In order to solve the above-mentioned technical problem, the invention discloses yak, ox and the introduces a collection of buffalo three in a kind of beef food
The detection primer of property composition, including primer GTB-F and primer GTB-R, wherein,
GTB-F:GAGGAGCCTGTTCTGTAATCG, its nucleotide sequence is as shown in SEQ ID NO.1；
GTB-R：ATGGCCTAATTCAACTAAGCACTC, its nucleotide sequence is as shown in SEQ ID NO.2.
The invention also discloses yak in a kind of beef food, the detection method of three kinds of derived components of ox and buffalo, bag
Include following steps：With the primer described in claim 1, quantitative pcr amplification is carried out to sample to be tested, obtains quantitative pcr amplification
Data；HRM data analyses detection amplification data is carried out using quantitative PCR apparatus software kit；
When expected amplification curve occurs in formation, show to contain yak, ox or buffalo composition in the testing sample；If
Expected amplification curve is occurred without, then without above-mentioned yak, ox or buffalo composition.
Further, the QPCR solution systems that described quantitative pcr amplification uses are as follows：HRM Mix 10 μ L, GTB-F
0.3 μ L, GTN-R0.3 μ L, Mg2+2 μ L, dd H2The μ L of O 5.4, the μ L of DNA profiling 2, above cumulative volume are 20 μ L.
Further, the QPCR reaction conditions that described quantitative pcr amplification uses are as follows：Pre-degeneration：95 DEG C of pre-degenerations
600s, heating rate are 4.4 DEG C/S, and period is 1 time；3 steps expand：95 DEG C of denaturation 10s, heating rate is 4.4 DEG C/S, 64->
55 DEG C of annealing 10s, heating rate are 2.2 DEG C/S, and 72 DEG C of extension 10s, heating rate is 4.4 DEG C/S, carries out 45 circulations altogether；It is high
Resolution ratio melts：95 DEG C, 60s, heating rate is 4.4 DEG C/S；40 DEG C, 60s, temperature rate is 2.2 DEG C/S；66 DEG C, 1s, rise
Warm speed is 2.2 DEG C/S；95 DEG C, 1s heating rates acquiescence；Period is 1 time；Cooling：37 DEG C of cooling 30s, rate of temperature fall are
1.0 DEG C/S, 1 circulation is carried out altogether.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention applies to high-resolution melting curve genotyping technique in three kinds of different calf-derived Cyclospora detections,
The reagent consumables cost of coherent detection is greatly reduced, while shortens detection time, and the degree of accuracy of detection can be improved, is avoided
False positive.With the market price, the same sample for detecting 500 batches, the cost of prior art is at 6000 yuans or so, and sheet
The cost of technology is 1800 yuans；The present invention needs only to a PCR system solution, and the operating time is the three of prior art
/ mono-.
2) superior technique is provided for three kinds of different calf-derived Cyclosporas in all kinds of beef foods of detection market sale
Ensure, and lower cost has using now high genetic test expense is reduced, and more conforms to customer demand.
Certainly, any product for implementing the present invention it is not absolutely required to reach all the above technique effect simultaneously.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, forms the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is the DNA HRM melting peakss curves of yak of the present invention, ox and buffalo；
Fig. 2 is the DNA HRM Genotyping curves of yak of the present invention, ox and buffalo.
Embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technical effect.
Yak in the beef food of embodiment 1, three kinds of derived components of ox and buffalo detection method structure：
1st, DNA is extracted
The DNA that selection conventional method (such as CTAB methods, SDS methods and commercial kit) is extracted in beef food to be measured is
Can.
2nd, specific primer synthesizes
A pair of the synthesis specific primer of the invention designed and developed：
GTB-F:GAGGAGCCTGTTCTGTAATCG, its nucleotide sequence is as shown in SEQ ID NO.1；
GTB-R：ATGGCCTAATTCAACTAAGCACTC, its nucleotide sequence is as shown in SEQ ID NO.2；
3rd, quantitative PCR solution system is prepared
Use HRM premix liquid kits (the Roche High Resolution Melting Mix of such as Roche Holding Ag
Kit QPCR solution, each component usage amount such as table 1 below) are prepared：
The QPCR solution systems of table 1
| Title | Volume (μ L) |
| HRM Mix | 10 |
| GTB-F | 0.3 |
| GTN-R | 0.3 |
| Mg2+ | 2 |
| dd H2O | 5.4 |
| DNA profiling | 2 |
| Cumulative volume | 20 |
4th, quantitative PCR reaction condition is set
HRM analyses are carried out using quantitative real time PCR Instrument, set gradually pre-degeneration, the amplification of 3 steps, high-resolution melts and drop
Warm four steps, design parameter such as table 2 below：
The QPCR reaction conditions of table 2
Pay attention to：The quantitative real time PCR Instrument (such as Roche Holding Ag LightCycle 96) with HRM functions must be used.
* represent：TouchDown cycle of annealings, since 64 DEG C, each cycle annealing temperature is reduced once, until 55
DEG C, 55 DEG C of annealing temperature is kept afterwards
5th, result judgement：
HRM data analyses are carried out using quantitative PCR apparatus software kit.With the DNA of yak, ox and buffalo as positive right
According to all there were significant differences for the HRM melting peakss curve (such as Fig. 1) and Genotyping curve (such as Fig. 2) of three, joins in this, as standard
According to curve.Unknown sample DNA carries out gained melting peakss curve and the standard of Genotyping curve and positive song after HRM analyses
Line carries out grouping comparison can determine that in unknown sample whether contain yak, ox or buffalo derived component.
It was found from Fig. 1 and Fig. 2, in Fig. 1 (melting peakss curve), the curve of three has obvious difference, can be had
The detection and identification of effect；In Fig. 2 (Genotyping curve), three has been distinctly divided into three different genotype, very good
Progress specific differentiate detection.For unknown sample, using yak, ox and buffalo as positive control, carry out simultaneously
HRM is analyzed.By the melting peakss curve of unknown sample and the melting peakss curve and Genotyping of Genotyping curve and positive control
Curve carries out packet comparison, and comparison result is consistent with the standard curve of any calf-derived Cyclospora, then containing this kind of ox source property into
Point；Otherwise, this kind of calf-derived Cyclospora is not contained.
The sensitivity experiment of embodiment 2
Yak, ox and the detection lower bound experiment of buffalo source property DNA solution concentration：By 10ng/ul yaks, ox and buffalo
Three kinds of DNA solutions are diluted to 1ng/ul, 0.1ng/ul, 0.01 ng/ul and 0.001ng/ul successively, and utilize inspection noted earlier
Survey technology is detected, final to determine that the present invention enter to yak, ox or the buffalo DNA solution that concentration is 0.01ng/ul
Row effective detection.
Mass fraction detects lower bound sensitivity experiment：Prepare yak meat gruel sample yak meat mass fraction point (yak meat
Amount/meat gruel gross mass) not Wei 1%, 0.1%, 0.01% and 0.001% 4 samples, with the art of this patent memory detect, it is right
The sample that mass fraction is 1%, 0.1% and 0.01% can be detected effectively, and the sample of 0.001% mass fraction can not be examined
Go out, therefore the present invention is 0.01% to the mass fraction detection lower bound of yak meat sample.After the same method, ox and buffalo source
Property composition mass fraction detection lower bound similarly be 0.01%.
The actual sample of embodiment 3 detects
From Sichuan Province Chengdu, Aba Prefecture, Deyang City etc., 10 cities and counties extract the batch of sample 80, wherein dried yak beef 43 batches
It is secondary, the batch of dried beef 37 (being collectively referred to as dried beef).The QPCR solution systems and amplification reaction condition established using this experiment are entered
Row HRM Genotypings detect, while are detected using concerned countries standard, then both testing results are compared, this reality
Proved recipe method testing result and related national standard testing result are completely the same, and accuracy rate is 100% (such as table 1)
The batch jerky food inspection result of 1 Sichuan Province of table 80
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification
And environment, and can be carried out in the scope of the invention is set forth herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in power appended by invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit requires.
Sequence table
<110>Design Inst. of Light Industry, Sichuan Prov.
<120>Yak, the detection primer and method of three kinds of derived components of ox and buffalo in a kind of beef food
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gaggagcctg ttctgtaatc g 21
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atggcctaat tcaactaagc actc 24
Claims (4)
1. yak, the detection primer of three kinds of derived components of ox and buffalo in a kind of beef food, it is characterised in that including primer
GTB-F and primer GTB-R, wherein,
GTB-F:GAGGAGCCTGTTCTGTAATCG, its nucleotide sequence is as shown in SEQ ID NO.1；
GTB-R：ATGGCCTAATTCAACTAAGCACTC, its nucleotide sequence is as shown in SEQ ID NO.2.
2. yak, the detection method of three kinds of derived components of ox and buffalo in a kind of beef food, it is characterised in that including following
Step：With the primer described in claim 1, quantitative pcr amplification is carried out to sample to be tested, obtains PCR amplification datas；Using calmly
Measure PCR instrument software kit and carry out HRM data analyses detection amplified production；
When expected amplification curve occurs in formation, show to contain yak, ox or buffalo composition in the testing sample；If do not go out
Amplification curve is now expected, then without above-mentioned yak, ox or buffalo composition.
3. detection method according to claim 2, it is characterised in that the QPCR solution that described quantitative pcr amplification uses
System is as follows：HRM Mix 10 μ L, GTB-F 0.3 μ L, GTN-R0.3 μ L, Mg2+2 μ L, dd H2The μ L of O 5.4, the μ L of DNA profiling 2,
Above cumulative volume is 20 μ L.
4. detection method according to claim 2, it is characterised in that the QPCR reactions that described quantitative pcr amplification uses
Condition is as follows：Pre-degeneration：95 DEG C of pre-degeneration 600s, heating rate are 4.4 DEG C/S, and period is 1 time；3 steps expand：95 DEG C of denaturation
10s, heating rate are 4.4 DEG C/S, 64->55 DEG C of annealing 10s, heating rate are 2.2 DEG C/S, and 72 DEG C extend 10s, heating rate
For 4.4 DEG C/S, 45 circulations are carried out altogether；High-resolution melts：95 DEG C, 60s, heating rate is 4.4 DEG C/S；40 DEG C, 60s, rise
Rate of temperature fall is 2.2 DEG C/S；66 DEG C, 1s, heating rate is 2.2 DEG C/S；95 DEG C, 1s heating rates acquiescence；Period is 1 time；
Cooling：37 DEG C of cooling 30s, rate of temperature fall is 1.0 DEG C/S, carries out 1 circulation altogether.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676864A (en) * | 2018-07-17 | 2018-10-19 | 安徽出入境检验检疫局检验检疫技术中心 | A kind of discrimination method of recombination beef |
| CN109457015A (en) * | 2018-12-29 | 2019-03-12 | 博奥生物集团有限公司 | Primer combines the application in Species estimation and/or the beef identification of ox |
| CN110106258A (en) * | 2019-05-14 | 2019-08-09 | 西南民族大学 | A kind of yak meat identification kit |
| CN114350822A (en) * | 2022-01-21 | 2022-04-15 | 锡林郭勒职业学院 | Primer and probe for synchronously detecting cattle, buffalo and yak in milk meat and quality control |
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| CN101712996A (en) * | 2009-12-14 | 2010-05-26 | 中国科学院昆明动物研究所 | Method for quickly identifying categories of meat and dried meat products of five domestic animals |
| CN101875976A (en) * | 2010-07-15 | 2010-11-03 | 中国农业科学院北京畜牧兽医研究所 | Triple PCR-RFLP method for identifying meat species source of yak jerky |
| CN104673900A (en) * | 2015-02-05 | 2015-06-03 | 中国肉类食品综合研究中心 | Method for identifying animal-derived ingredients in meat or meat product |
| CN106755341A (en) * | 2016-11-30 | 2017-05-31 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of yak and Carnis Bovis seu Bubali source property mirror method for distinguishing |
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Patent Citations (4)
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| CN101712996A (en) * | 2009-12-14 | 2010-05-26 | 中国科学院昆明动物研究所 | Method for quickly identifying categories of meat and dried meat products of five domestic animals |
| CN101875976A (en) * | 2010-07-15 | 2010-11-03 | 中国农业科学院北京畜牧兽医研究所 | Triple PCR-RFLP method for identifying meat species source of yak jerky |
| CN104673900A (en) * | 2015-02-05 | 2015-06-03 | 中国肉类食品综合研究中心 | Method for identifying animal-derived ingredients in meat or meat product |
| CN106755341A (en) * | 2016-11-30 | 2017-05-31 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of yak and Carnis Bovis seu Bubali source property mirror method for distinguishing |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676864A (en) * | 2018-07-17 | 2018-10-19 | 安徽出入境检验检疫局检验检疫技术中心 | A kind of discrimination method of recombination beef |
| CN109457015A (en) * | 2018-12-29 | 2019-03-12 | 博奥生物集团有限公司 | Primer combines the application in Species estimation and/or the beef identification of ox |
| CN110106258A (en) * | 2019-05-14 | 2019-08-09 | 西南民族大学 | A kind of yak meat identification kit |
| CN110106258B (en) * | 2019-05-14 | 2022-09-23 | 西南民族大学 | Yak meat identification kit |
| CN114350822A (en) * | 2022-01-21 | 2022-04-15 | 锡林郭勒职业学院 | Primer and probe for synchronously detecting cattle, buffalo and yak in milk meat and quality control |
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