CN108823318A - The kit and detection method of bovine material in a kind of detection food - Google Patents
The kit and detection method of bovine material in a kind of detection food Download PDFInfo
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- CN108823318A CN108823318A CN201710317229.1A CN201710317229A CN108823318A CN 108823318 A CN108823318 A CN 108823318A CN 201710317229 A CN201710317229 A CN 201710317229A CN 108823318 A CN108823318 A CN 108823318A
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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
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Abstract
The present invention provides a kind of gene order for for detecting calf-derived Cyclospora, the sequence as shown in SEQ ID NO.1 and 2, as shown in SEQ ID NO.3 and 4, as shown in SEQ ID NO.5 and 6；As shown in SEQ ID NO.7 and 8 or the primer as shown in SEQ ID NO.9 and 10.It can be very good to distinguish the specific ingredient in meat products using these primer pairs.
Description
Technical field
The invention belongs to field of food detection and technical field of molecular biology, specifically, how belong to using real-time
Fluorescence RAA technology and combine special primer detection food in whether the method containing bovine material.
Background technique
In recent years, food-safety problem, masses' reflection is more more and more intense, the degree of social concern is also higher and higher.2016 3
The moon-April, 8 batch beef ball with soup inside that Chengdu food and medicine Inspection Research institute produces HighPower king Food Co., Ltd into
Row sampling observation, it is as a result all unqualified：Part batch beef dumplings without beef, part batch beef dumplings detected duck ingredient and pig at
Point.Then, city quality supervision and control office law enfrocement official has carried out examination of law enforcement to the said firm according to unqualified survey report, and
Related punishment is carried out.Some the food enterprises are used inferior materials and turned out substandard goods for oneself private interests, are adulterated, and consumption is seriously compromised
The interests of person and the life and health of people.Generally for raw beef product, we can pass through color, grain of meat and taste etc.
It is identified, but we are difficult to be identified with this conventional method for processed cooked meat product.
Recombinase-mediated amplification (Recombinase-aid Amplification, RAA) technology be also it is a kind of at a constant temperature
It can make the method for nucleic acid rapid amplifying.Unlike RPA, RAA amplification, which is used, to be obtained from bacterium or fungi
Recombinase, under 37 DEG C of constant temperature, which can combine closely with primed DNA, the condensate of enzyme and primer be formed, when primer exists
When searching the sequence of complete complementary therewith on template DNA, in single-stranded DNA binding protein (single-strandedDNA
Binding, SSB) with the help of, make template DNA unwinding, and under the action of archaeal dna polymerase, forms new DNA complementary strand, instead
Answering product is also to be increased with exponential, can obtain that the amplification piece that agarose gel electrophoresis detects can be used usually in 1h
Section.Fluorophor is added in RAA reaction system, monitors entire RAA amplification procedure in real time using the accumulation of fluorescence signal, 20 points
, it can be achieved that quantitative and qualitative analysis to starting template in clock.Entirely react simple and quick, because not needing high temperature circulation, institute
To be particularly suitable for using in the non-laboratory testing place for there are a large amount of samples, it is suitable for field of rapid food detection.
Summary of the invention
Present invention aims at, provide one group it is preferred after detection food in calf-derived Cyclospora RAA primer pair and one
The fluorescence probe to match.
In some preferred modes, the present invention is provided to detect the gene order of calf-derived Cyclospora, the sequence such as SEQ
Shown in ID NO.1 and 2, as shown in SEQ ID NO.3 and 4, as shown in SEQ ID NO.5 and 6；As shown in SEQ ID NO.7 and 8
Or the primer as shown in SEQ ID NO.9 and 10.Preferably, these primers detect calf-derived Cyclospora using RAA technology.
A second object of the present invention is to provide one kind to contain above-mentioned primer sets and fluorescence probe, for detecting ox in food
The kit of derived components.In some specific modes, the kit include the necessary reagent of RAA and selected from following a pair of or
The multipair primer sequence of person：Shown in SEQ ID NO.1 and 2, as shown in SEQ ID NO.3 and 4, such as the institute of SEQ ID NO.5 and 6
Show；As shown in SEQ ID NO.7 and 8 or the primer as shown in SEQ ID NO.9 and 10.
Fourth object of the present invention is to detect to carry out real time fluorescent quantitative RAA and adulterate containing for bovine material in food
Amount provides primer pair, these primer pairs are selected from the following primer pair：Shown in SEQ ID NO.1 and 2, such as SEQ ID NO.3 and 4
It is shown, as shown in SEQ ID NO.5 and 6；As shown in SEQ ID NO.7 and 8 or the primer as shown in SEQ ID NO.9 and 10.
Technical solution of the present invention is summarized as follows：The RAA primer sets of bovine material in a kind of detection food, wherein this draws
Object is to a pair of or several right in following primer pair：No. 1 primer pair, No. 2 primer pairs, No. 3 primer pairs, No. 4 primer pairs or 5
Number primer pair composition, the nucleotide sequence of No. 1 primer pair is respectively as shown in SEQ ID NO.1 and 2；No. 2 primer pairs
Nucleotide sequence respectively as shown in SEQ ID NO.3 and 4；The nucleotide sequence of No. 3 primer pairs is respectively such as SEQ ID
Shown in NO.5 and 6；The nucleotide sequence of No. 4 primer pairs is respectively as shown in SEQ ID NO.7 and 8；No. 5 primer pairs
Nucleotide sequence is respectively as shown in SEQ ID NO.9 and 10.
In some preferred embodiments, locating primer pair is the sequence as shown in SEQ ID NO.1 and 2.
The kit of bovine material in a kind of detection food, including primer pair, RAA powdered reagent, A Buffer, B
Buffer, sterile water, the primer pair are a pair of or several right in following primer pair：No. 1 primer pair, No. 2 primer pairs, 3
Number primer pair, No. 4 primer pairs or No. 5 primer pairs, wherein No. 1 primer pair nucleotide sequence is respectively such as the institute of SEQ ID NO.1 and 2
Show, the nucleotide sequence of No. 2 primer pairs is respectively as shown in SEQ ID NO.3 and 4, the nucleotides sequence of No. 3 primer pairs
Column are respectively as shown in SEQ ID NO.5 and 6, and the nucleic acid sequence of No. 4 primer pairs is respectively as shown in SEQ ID NO.7 and 8, institute
The nucleotide sequences of No. 5 primer pairs is stated respectively as shown in SEQ ID NO.9 and 10.In some preferred embodiments, locating
Primer pair be the sequence as shown in SEQ ID NO.1 and 2.
The A Buffer is 20%PEG；B Buffer is 280mM MgAc.
The ingredient of the RAA powdered reagent is as follows：1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L
RecA recombinates zymoprotein (SC-recA/BS-recA) or 30ng/ μ L Rad51,30ng/ μ L Bsu archaeal dna polymerase, 100mmol/
L Tricine, 20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/ μ L creatine kinase.Alternatively, the component of RAA powdered reagent is such as
Under：1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA recombination zymoprotein (SC-recA/BS-recA) or
30ng/ μ L Rad51,30ng/ μ L Bsu archaeal dna polymerase, 30ng/ μ L Exo exonuclease, 100mmol/L Tricine,
20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/ μ L creatine kinase.
In some preferred embodiments, the target fragment size of primer sets amplification of the invention is as follows, No. 1 primer pair：
173bp；No. 2 primer pairs：179bp；No. 3 primer pairs：214bp；No. 4 primer pairs：233bp；No. 5 primer pairs：273bp.
Whether primer pair of the invention not only can also can specifically be distinguished from food and be contained with specific detection bovine material
The ingredients such as beef have good detection and identification effect especially for false food is joined.
Beneficial effect
Kit of the invention can quickly, accurately, sensitively detect the bovine material in meat products.Especially for line
The serious deep processing meat products of mitochondrial DNA degradation, the target fragment because providing primer is small fragment, can be very good to be examined
It surveys, and then it is excessive to overcome target fragment, the case where can not be detected for cooked meat product in actually detected.
Detailed description of the invention
Fig. 1 is 5 pairs of primers involved in the present invention, i.e. No. 1 primer pair is respectively to the meat products genomic DNA of 5 species
Carry out RAA amplification as a result, M be DL 2000DNA Marker (from the bottom up, 100bp, 250bp, 500bp, 750bp,
1000bp,2000bp)；1-5 is respectively the genomic DNA of chicken, pig, duck, ox, sheep, and 6 be negative control, and 7 be positive control.
Fig. 2 is the testing result figure of cow genome various concentration.The best primer sensitivity of ox (No. 1 primer pair)；1-5 is successively
For：189ng/uL,18.9ng/uL,1.89ng/uL,189pg/uL,18.9pg/uL；6 be negative control, and 7 be positive control；
Fig. 3 A-3E is the real-time of commercially available beef product genomic DNA of No. 1 primer pair through deep processing involved in the present invention
Fluorescence RAA amplification；Fig. 3 A is beef and negative control, and Fig. 3 B is beef and chicken Parallel testing；Fig. 3 C is beef and pig
Meat Parallel testing；Fig. 3 D is beef and duck Parallel testing；Fig. 3 E is beef and mutton Parallel testing.
Fig. 4 be RAA expand sensitivity result figure, 1-3 be ox genomic DNA concentration (be followed successively by 18.9ng/ μ L,
1.89ng/ μ L, 189pg/ μ L), 4 be negative control.
If the testing result figure for the beef product that Fig. 5 is deep processing, 1 is positive control, 2,3 be the beef product of deep processing
Genomic DNA result (cold cuts), 4 be negative control.
Specific implementation method
Below by way of specific embodiment, the present invention is further described.
Embodiment 1：Design of primers
First from NCBI (National Center for Biotechnology Information, American National biology
Technology information centre) Genbank database in find the mitochondrial DNA complete sequence of ox and chicken, the mitochondrial DNA total order of pig
Column, duck mtDNA complete sequence, sheep mitochondrial DNA complete sequence (the ox Bos taurus number of logging in：AY676864.1；Chicken
Gallus gallus accession number：KM096864；The pig Susscrofa domesticus number of logging in：NC_012095.1；Duck Anas
Platyrhynchos accession number：KJ833587.1；Sheep Ovis aries accession number：KR868678.1；), carry out sequence alignment.
According to comparison result, cytochrome b (cytb) gene order (KT151960.1) encoded using ox mitochondrial DNA
It is put into Primer5.0 and carries out design of primers, obtain multipair different primer pair and carry out screening test.
With reference to ox (Bos Taurus) and chicken (Gallus gallus), pig (Sus scrofa while design primer
Domesticus), the sequence comparison of duck (Anas platyrhynchos), sheep (Ovis aries) guarantees to design
Primer sequence for expanding bovine mitochondrial gene group segment is not matched with porcine mtdna genome arbitrary sequence.It considers simultaneously
The genomic DNA degradation of cooked meat product is obvious, thus set primer range is 100bp-400bp.And in order to enhance primer
Specificity, the G/C content of set primer is between 30%-50%.
It according to conditions above, picks out 300 pairs of bovine material primers and synthesizes, while with chicken, pig, duck, ox, the full base of sheep
Because group DNA is template, primer specificity and product amount are examined through standard PCR amplification, it is as follows to select optimal primer pair the selection result,
5 pairs of primers i.e. provided by the present invention are used as RAA amplification experiment.No. 1 primer pair：
F：5'-ATTTCGGTTCCCTCCTGGGAATCTGCCTAATC-3'(SEQ ID NO.1)
R：5'-CATTGAAGCTCCGTTTGCGTGTATGTATCGG-3'(SEQ ID NO.2)
No. 2 primer pairs：
F：5'-TCATCGACCTTCCAGCCCCATCAAACATTTCA-3'(SEQ ID NO.3)
R：5'-TCAGCCGTAGTTCACGTCTCGGCAGATATG-3'(SEQ ID NO.4)
No. 3 primer pairs：
F：5'-CTAGCAATACACTACACATCCGACACAACA-3'(SEQ ID NO.5)
R：5'-TGAGCAGAAGGATTACTCCAATATTTCATG-3'(SEQ ID NO.6)
No. 4 primer pairs：
F：5'-ATTCTCAGTAGACAAAGCAACCCTTACCCG-3'(SEQ ID NO.7)
R：5'-ATAGTACTAGTAGTATTAGAGCTAGAATTAG-3'(SEQ ID NO.8)
No. 5 primer pairs：
F：5'-ATAGTCCACCTACTATTCCTCCACGAAACAG-3'(SEQ ID NO.9)
R：5'-GATTGATCGTAAGATTGCGTATGCAAATAAG-3'(SEQ ID NO.10)
Examples of implementation 2：The screening (specificity and sensitivity verifying) of best primer pair
No. 1 primer pair：
Extract respectively chicken, pig, duck, ox, 5 species of sheep fresh meat tissue in genomic DNA, the method for extraction is as follows：
By sample (such as：Green meat sample) it is processed into minced meat shape；30mg is taken to be added in 1.5mlEP pipe；500 μ l are added to split
Solve liquid (1M tris-Hcl PH8.0；0.5M EDTA PH8.0；5M Nacl；0.5%N-Lanroyl Sarc0sine (the N- month
Osmanthus acylsarcosine)；Proteinase K (20mg/ μ l)；70 DEG C of RNaseA (10mg/ μ l) 0.5-1h or 50 DEG C of digestion are digested overnight, and are centrifuged
(12000r/min, 5-15min), takes supernatant；In supernatant plus isometric phenol, chloroform and isoamyl alcohol (25: 24: 1) extract；
400 μ l water phases are taken, adds 40 μ lNacl, 1ml ice ethyl alcohol, places 30min on ice；It is centrifuged (12000r/min, 5min), abandons supernatant；
It is washed 3 times, is dried with 1ml70% dehydrated alcohol；Add 70-100 μ lTE, stands 30min.Mentioned genomic DNA is placed in -20 DEG C of ice
It saves in case, is used for experiment later.RAA reaction is carried out as template.
RAA reacts dry powder：1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA recombinate zymoprotein (SC-
RecA/BS-recA) or 30ng/ μ L Rad51,30ng/ μ L Bsu archaeal dna polymerase, 100mmol/L Tricine, 20%PEG,
5mmol/L dithiothreitol (DTT), 100ng/ μ L creatine kinase.
Reaction system：50μl：
The template of the gene DNA of extraction：2.0μl；The A Buffer of 20%PEG：12.5μl；The B of 280mM MgAc
Buffer：2.5μl；Upstream and downstream primer distinguishes 2.0 μ l；ddH2O 31 μ l and RAA react powdered reagent.
The configuration method of 50 μ l reaction systems：The template of the gene DNA of extraction is added in upper RAA reaction powdered reagent：
2.0μl；The A Buffer of 20%PEG：12.5μl；The B Buffer of 280mM MgAc：2.5μl；Upstream and downstream primer distinguishes 2.0 μ
l；ddH2O31 μ l, the reaction being then uniformly mixed into following steps expand.
Reaction condition：：It is reacted 30 minutes at 37 DEG C.
Agar electrophoresis is carried out after reaction result, as a result as shown in Figure 1,1-5 be chicken, pig, duck, ox, sheep genomic DNA, 6
It is positive control for negative control, 7；
As shown in Fig. 2, 1-5 be ox genomic DNA concentration (be followed successively by 189ng/ μ L, 18.9ng/ μ L, 1.89ng/ μ L,
189pg/ μ L, 18.9pg/ μ L) amplification, 6 be negative control, and 7 be positive control.M be DL 2000DNA Marker (from
Under up, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp).
It is the result figure of No. 1 primer above, it can be seen that No. 1 primer can distinguish different meat sources, have preferable
Specificity, merely for beef product, when DNA concentration be 189ng/uL, 18.9ng/uL, 1.89ng/uL when,
Testing result can be obtained, and other concentration illustrate that testing result is not obvious enough without the appearance of apparent lines.
Same system and method is taken to detect other 4 pairs of primers, it is as a result as follows：
No. 2 primer pairs：Sensitivity is bad, the DNA of low concentration, and amplification efficiency is not high, it may be possible to because of the matching degree of sequence
It is not high.
No. 3 primer pairs：The result of No. 3 primers is similar to the result of No. 1 primer, and specific data are omited.
No. 4 primer pairs：The result of No. 3 primers is similar to the result of No. 1 primer, and specific data are omited.
No. 5 primer pairs：Specificity is bad, cannot distinguish a species very well.
From the point of view of result above, 1,3, No. 4 primer pair is optimal primer pair, is suitble to be expanded with the method for RAA, have
There are good specificity and sensitivity.
The above sample is the experiment and screening that fresh meat product are done, when select cooked meat product (it is edible, 9 it is mature or
The mature meat of person 10 is cooked similar RAA amplification experiment same as described above, and specific data and process are omited), it has been found that, 1,3, No. 5 is drawn
Object is to good expanding effect.This demonstrate that but there is different special primers for fresh or cooked food beef,
Reason may be that some changes have occurred in cooked DNA shape, to influence the amplification of sequence, it is also possible to fresh and cold cuts
The ingredient of product or interference are different, but have opposite for same sequence as a result, specific mechanism is unclear.
Examples of implementation 3：ABI7500 real-time fluorescence RAA amplification verifying (by taking No. 1 primer pair as an example)
The sequence of corresponding probe：
CACTACACATCCGACACAACAACAGCATTC[FAM-dT]C[THF][BHQ-dT]
CTGTTACCCATATCTG(SEQ ID NO.11)
Reaction system：50μl：
The template (fresh sample, method are identical as examples of implementation 2) (chicken, pig, duck, ox, sheep) of the gene DNA of extraction is respectively
It is：2.0μl；The A Buffer of 20%PEG：12.5μl；The B Buffer of 280mM MgAc：2.5μl；Upstream and downstream primer difference
2.0μl；0.6 μ l of Probe (probe)；ddH2O 28.4 μ l and real-time fluorescence RAA react powdered reagent.
Real-time fluorescence RAA reacts dry powder：1mmol/L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA recombinase
Albumen (SC-recA/BS-recA) or 30ng/ μ L Rad51,30ng/ μ L Bsu archaeal dna polymerase, outside 30ng/ μ L Exo nucleic acid
Enzyme cutting, 100mmol/L Tricine, 20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/cL creatine kinase.
The configuration method of 50 μ l reaction systems：The gene of extraction is added in above-mentioned real-time fluorescence RAA reaction powdered reagent
The template of DNA：2.0μl；The A Buffer of 20%PEG：12.5μl；The B Buffer of 280mM MgAc：2.5μl；Upstream and downstream is drawn
Object distinguishes 2.0 μ l；0.6 μ l of probe (Probe)；ddH228.4 μ l of O, the reaction being then uniformly mixed into following steps expand.
It first turns on ABI7500 real-time fluorescence amplification instrument (Applied biosystems) and carries out 20-30min preheating,
Design following procedure：Holding stage, 39 DEG C of 60s, 1 circulation；Cycling stage, 39 DEG C of 30s, 40 circulations, and
Collect fluorescent signal data.
As a result：Finally, analyzing amplification, as that illustrated in figures 3 a-d, Fig. 3 A is beef and negative control, and Fig. 3 B is
Beef and chicken Parallel testing；Fig. 3 C is beef and pork Parallel testing；Fig. 3 D is beef and duck Parallel testing；Fig. 3 E is ox
Meat and mutton Parallel testing；Then illustrate to detect meat products using primer pair, can with specific detection beef product, without
The detection of inaccuracy is caused to the interference of other meat products.
When using the detection of DNA concentration, as shown in figure 4, the genomic DNA concentration that 1-3 is ox (is followed successively by
18.9ng/ μ L, 1.89ng/ μ L, 189pg/ μ L), 4 be negative control.
It is as a result similar with No. 1 primer pair simultaneously using similar ABI7500 real-time fluorescence RAA amplification verifying primer pair 3 and 4,
Specific data are omited.
Examples of implementation 4：The commercially available beef product through deep processing (cooked) is verified (by taking No. 1 primer pair as an example)
1, the genomic DNA of the beef product of deep processing is extracted：Method is the same as the genomic DNA for mentioning meat products in embodiment 1
It is identical.
2, system：50 μ l (templates：2.0μl；A Buffer 12.5μl；B Buffer 2.5μl；Upstream and downstream primer difference
2.0μl；Probe 0.6μl；ddH2O 28.4μl).Each component concrete composition is as in Example 3.
3, ABI7500 amplified fluorescence is verified
Genomic DNA mentioned above is template, carries out real-time fluorescence RAA amplification with primer 1 in the present invention.
Reaction condition：
The first step：39℃60s；
Second step：39 DEG C of 30s, 40 circulations.
Finally, analyzing amplification, as shown in figure 5,1 is negative control, 2-3 is the beef product of deep processing
Genome, 4 be positive control.
Finally, sequencing or following sequence are carried out to the 2-3 product of amplification：
The upstream ATTTCGGTTCCCTCCTGGGAATCTGCCTAATC)
CTACAAATCCTCACAGGCCTATTCCTAGCAATACACTACACATCCGACACAACAACAGCATTCTCCTCTGTTACCCA
TATCTGCCGAGACGTGAACTACGGCTGAATCATCCGATACATACACGCAAACGGAG CTTCAATG (downstream) (SEQ ID
NO.12, colored is the sequence of primer pair 1).Pass through the cytochrome b with the ox mitochondrial DNA coding in embodiment 1
(cytb) gene order (KT151960.1-1140bp) compares, and discovery has 100% matching degree with one section therein.
, that is to say, bright, which is the distinguished sequence of species differentiation for this.
When the amplification for carrying out other primer pairs using same method (is contaminated with the meat of other animals in beef product
Product, chicken, duck, pork), it has been found that 3, No. 5 primer pairs have good expanding effect, have a significant specificity, and No. 4
Primer cannot but detect well, and when having the mixing of other meat products, not have specificity but.This explanation, 1
Number primer all has good discrimination, specificity with higher and sensitivity for fresh or shortening meat product.Meanwhile
No. 5 primers have good differentiation for cooked beef, and cannot distinguish to fresh meat product.
In the case where lacking any element specifically disclosed herein, limitation, may be implemented illustrated and described herein
Invention.Used terms and expressions method is used as the term of explanation rather than limits, and is not intended in these terms and table
Up to any equivalent for excluding shown and described feature or part thereof in the use of method, and it should be realized that various remodeling exist
It is all feasible in the scope of the present invention.It is therefore to be understood that although specifically being disclosed by various embodiments and optional feature
The present invention, but the modifications and variations of concept as described herein can be used by those of ordinary skill in the art, and recognize
It is fallen into for these modifications and variations within the scope of the present invention of the appended claims restriction.
It is described herein or record article, patent, patent application and every other document and can electronically obtain
The content of information to a certain extent in full include herein by reference, just as each individual publication by specific and single
Solely point out by reference.Applicant retains from any of any this article, patent, patent application or other documents
And all material and information are incorporated into the right in the application.
Claims (9)
1. a kind of for detecting the gene order of calf-derived Cyclospora, the sequence is as shown in SEQ ID NO.1 and 2, such as SEQ ID
Shown in NO.3 and 4, as shown in SEQ ID NO.5 and 6；As shown in SEQ ID NO.7 and 8 or as shown in SEQ ID NO.9 and 10
Primer.
2. the kit of bovine material in a kind of detection food；The kit includes the necessary reagent of RAA and is selected from as next
To or multipair primer sequence：SEQ ID NO.1 and 2 is shown, such as SEQ ID NO.3 and 4 is shown, such as SEQ ID NO.5 and 6
It is shown；As shown in SEQ ID NO.7 and 8 or the primer as shown in SEQ ID NO.9 and 10.
3. a kind of progress real time fluorescent quantitative RAA, detects the kit for adulterating bovine material content in food, which includes
Primer pair, these primer pairs are a pair of or multipair in following primer pair：Shown in SEQ ID NO.1 and 2, such as SEQ ID
Shown in NO.3 and 4, as shown in SEQ ID NO.5 and 6；As shown in SEQ ID NO.7 and 8 or as shown in SEQ ID NO.9 and 10
Primer.
4. primer pair：Shown in SEQ ID NO.1 and 2, shown in SEQ ID NO.3 and 4, shown in SEQ ID NO.5 and 6；SEQ ID
Shown in NO.7 and 8 or the primer as shown in SEQ ID NO.9 and 10 mixes the purposes of bovine material in detection food.
5. gene order described in one of -4, kit or purposes according to claim 1, wherein locating primer pair is such as SEQ
Sequence shown in ID NO.1 and 2.
6. kit according to claim 2 or 3, wherein further include A Buffer, B Buffer and the examination of RAA dry powder
Agent.
7. kit according to claim 6, wherein the Dun A Buffer is 20%PEG；B Buffer is 280mM
MgAc。
8. kit according to claim 7, wherein the ingredient of the RAA powdered reagent is as follows：1mmol/L
DNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA recombination zymoprotein (SC-recA/BS-recA) or 30ng/ μ L Rad51,
30ng/ μ L Bsu archaeal dna polymerase, 100mmol/L Tricine, 20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/ μ L flesh
Acid kinase.
9. kit according to claim 8, wherein further include probe sequence be SEQ ID NO.11 shown in sequence,
With Exo exonuclease.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708682A (en) * | 2021-02-08 | 2021-04-27 | 韩山师范学院 | Primer pair and probe for detecting bovine-derived components, kit and application thereof |
| CN117286262A (en) * | 2023-11-24 | 2023-12-26 | 中国农业科学院北京畜牧兽医研究所 | Fluorescent PCR detection kit and method for detecting authenticity of dairy product by using same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293899A (en) * | 2013-07-19 | 2015-01-21 | 聂凌云 | Method for detecting horsemeat in food |
| CN105274246A (en) * | 2015-11-20 | 2016-01-27 | 华中农业大学 | Reagent kit for bovine-derived component identification and detection of multi-species provenance components in products of bovine-derived components |
-
2017
- 2017-05-03 CN CN201710317229.1A patent/CN108823318A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293899A (en) * | 2013-07-19 | 2015-01-21 | 聂凌云 | Method for detecting horsemeat in food |
| CN105274246A (en) * | 2015-11-20 | 2016-01-27 | 华中农业大学 | Reagent kit for bovine-derived component identification and detection of multi-species provenance components in products of bovine-derived components |
Non-Patent Citations (3)
| Title |
|---|
| ZHANG CHUNLAI等: "A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses" * |
| 冯涛等: "动物源性成分交叉引物等温扩增快速检测方法的研究和建立" * |
| 吕蓓等: "用重组酶介导扩增技术快速扩增核酸" * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708682A (en) * | 2021-02-08 | 2021-04-27 | 韩山师范学院 | Primer pair and probe for detecting bovine-derived components, kit and application thereof |
| CN112708682B (en) * | 2021-02-08 | 2024-02-02 | 韩山师范学院 | Primer pair and probe for detecting bovine-derived components, kit and application thereof |
| CN117286262A (en) * | 2023-11-24 | 2023-12-26 | 中国农业科学院北京畜牧兽医研究所 | Fluorescent PCR detection kit and method for detecting authenticity of dairy product by using same |
| CN117286262B (en) * | 2023-11-24 | 2024-03-19 | 中国农业科学院北京畜牧兽医研究所 | Fluorescent PCR detection kit and method for detecting authenticity of dairy product by using same |
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