CN103966346B - A kind of detect catfish kind method and detection kit and primer - Google Patents
A kind of detect catfish kind method and detection kit and primer Download PDFInfo
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Abstract
The invention discloses a kind of detect catfish kind method and detection kit and primer.The method comprises the following steps: (1) with the genomic dna of sample to be detected for template, with the genomic dna of specificity amplification primer to amplification sample to be detected, the right sequence of this specificity amplification primer is respectively as shown in SEQ ID NO:1 ~ SEQ ID NO:12 in sequence table, (2) gained pcr amplification product is carried out gel electrophoresis analysis, have the sample to be tested of specific band to be clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus).Compare with traditional method, the method has accuracy and uniqueness.Operator do not need a large amount of working experiences, only need test according to operation steps.It is high that test kit of the present invention has required detection time short detected result specificity, the simple advantage of detected result decision method.
Description
Technical field
The present invention relates to fish detection field, be specifically related to a kind of detect catfish kind method and detection kit and primer.
Background technology
Catfish is global fish, is distributed widely in the whole North Atlantic and waters, North Pacific.Waters, East Coast of the United States of America from Maine to North Carolina produces Atlantic Ocean catfish (Clupea harengus).Therefrom california produces clupea pallasi (Clupea pallasi) to the U.S.West Coast waters of Alaska Bering Sea.The resource of Atlantic Ocean catfish and clupea pallasi is all very abundant.Catfish has very high pharmaceutical use, and its spermary contains abundant protamine, deoxynucleotide and arginine, is to extract the important source material making protamine injection liquid and insulinum protaminatum cumzinco.
Catfish is raw material sources prepared by protamine.At present, mainly adopt traditional morphology differential method for fish mirror method for distinguishing, namely morphological specificity comparison is carried out to complete fish body, but this authentication method greatest drawback is the fish body that Structure of need is complete, can not damage be had, and complicated operation, strongly professional.If only with fish tissues sample for material, adopt traditional entirety morphology means, then cannot carry out fish discriminating.
Summary of the invention
The technical problem to be solved in the present invention is that overcoming traditional catfish traditional form differential method needs complete fish body to carry out morphological specificity comparison, the fish body that Structure of need is complete, can not have damage, traditional catfish morphology differential method is made to have complicated operation, strongly professional defect.Adopt detection method of the present invention to detect the kind of catfish, required detection time is short, and cost is low, and detected result specificity is high, and detected result decision method is simple.
For solving the problems of the technologies described above, one of technical scheme of the present invention is: a kind of method detecting catfish kind, said method comprising the steps of: (1) increase the genomic dna of sample to be detected, the right sequence of described specificity amplification primer is respectively as SEQ ID NO:1 in sequence table and SEQ ID NO:2, or SEQ IDNO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8, or SEQ ID NO:9 and SEQ ID NO:10, or shown in SEQ ID NO:11 and SEQ IDNO:12
(2) step (1) gained pcr amplification product is carried out gel electrophoresis analysis, have the sample to be tested of specific band to be clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus).
According to the present invention, sample to be tested described in step (1) is the sample to be tested of this area routine, is preferably a part for fish body to be measured, preferably the flesh of fish, internal organ, fish head, the fish sperm, one or more in roe and fish scale.
The method extracting the genomic dna of sample to be detected described in step (1) is this area ordinary method, and described method preferably comprises the following steps:
Get fish tissues sample to add liquid nitrogen and to mill 10min, or 100 DEG C are dried 5-8 hour, mill after to be dried 10min.
Get the sample after pulverizing and add EP pipe, add cell pyrolysis liquid 1ml, add Proteinase K 20 μ l, mixing.
At the constant water bath box water-bath 10-40min of 60-70 DEG C, also can at 30-40 DEG C of water-bath 12-24h, interval concussion EP pipe is for several times.
After cracked, desk centrifuge, with the centrifugal 5-10min of 12000-13000rpm, is got supernatant liquor and is carefully sucked in another centrifuge tube.
Add 2 times of volume isopropanol, after reversing mixing, can filament be seen, choose with suction nozzle, dry.
Add 200 μ lTE and heavily wash dissolving, add the phenol of equivalent: chloroform: primary isoamyl alcohol (25:24:1) concussion mixing, the centrifugal 5-10min of rotating speed 12000-13000rpm.
Get upper solution to manage to another, add isopyknic chloroform: primary isoamyl alcohol (24:1), concussion mixing, the centrifugal 5-10min of rotating speed 12000-13000rpm.
Get upper solution to manage to another, add the ammonium acetate of the 7.5mol/L of 1/2 volume, then add the dehydrated alcohol of 2 times of volumes, after mixing, the centrifugal 5-10min of rotating speed 12000-13000rpm.
Carefully pour out supernatant liquor, tube wall residual droplets is removed by thieving paper.
With washing with alcohol throw out 2-3 time of 1ml70%, the centrifugal 5-10min of rotating speed 12000-13000rpm.
Carefully outwell supernatant liquor, EP pipe is put 37 DEG C, baking oven and is dried 3-5h.
The system that PCR described in step (1) reacts is preferably 1 × PCR reaction buffer, 10 ~ 15mmol/L Mg
2+, 0.2 ~ 0.3mmol/L dNTP, 0.1 ~ 0.3 μM of specificity amplification primer pair, 0.01 ~ 0.1U/ μ LTaq enzyme, 10 ~ 100ng/ μ LDNA template.Be more preferably: 1 × PCR reaction buffer, 12.5mmol/LMg
2+, 0.25mmol/LdNTP, 0.2 μM of specificity amplification primer pair, 0.04U/ μ LTaq enzyme and 50ng/ μ L DNA profiling.
The amplification program that PCR described in step (1) reacts is preferably: (1) 93 ~ 95 DEG C of denaturation 2 ~ 4min, (2) 93 ~ 95 DEG C of sex change 28 ~ 32s, (3) 56 ~ 60 DEG C of annealing 28 ~ 32s, (4) 72 DEG C extend 1 ~ 2min, wherein step (2) ~ (4) cycle index 30 ~ 35 times, (5) 72 DEG C extend 3 ~ 5min, (6) 4 DEG C of termination reactions.Be more preferably: (1) 94 DEG C of denaturation 3min, (2) 94 DEG C of sex change 30s, (3) 58 DEG C of annealing 30s, (4) 72 DEG C extend 1min, wherein step (2) ~ (4) cycle index 30 times, (5) 72 DEG C extend 4min, (6) 4 DEG C of termination reactions.
Method of the present invention more preferably also comprises step (3): checked order by step (1) gained pcr amplification product, the COI gene that sequencing result and catfish belong to is carried out sequence alignment, if COI gene matching degree >=99% of sample sequence and clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus) is clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus).
Matching degree between gene is represented by Ident value, and Ident value refers to the matching degree of two sections of nucleotide sequences, and this value, more close to 100%, illustrates that the similarity of two nucleotide sequences is higher.Consider the accuracy impact of gene amplification and order-checking, when Ident value >=99%, just can judge that the gene of species to be measured and goal gene are from same species.Therefore when the COI gene belonged to when sequencing result and catfish carries out sequence alignment, if Ident value >=99% of sample sequence and clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus) COI gene, then determine that sample to be tested is clupea pallasi or Atlantic Ocean catfish.
Order-checking described in step (3) and sequence alignment method are this area ordinary method, the method of wherein said order-checking is preferably for utilizing nucleic acid sequencing instrument to check order, and the method for described sequence alignment is preferably for utilizing Blast method to carry out sequence alignment.
For solving the problems of the technologies described above, two of technical scheme of the present invention is: a kind of test kit detecting catfish kind, it comprises specificity amplification primer pair, the right sequence of described specificity amplification primer is respectively as SEQ ID NO:1 in sequence table and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ IDNO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8, or SEQ ID NO:9 and SEQ ID NO:10, or shown in SEQ ID NO:11 and SEQ ID NO:12.
Test kit of the present invention preferably also comprises 10 × PCR damping fluid, Taq enzyme, dNTP solution and MgCl
2.Described 10 × PCR damping fluid, Taq enzyme, dNTP solution and MgCl
2all as described in the routine of this area.
For solving the problems of the technologies described above, three of technical scheme of the present invention is: a kind of primer detecting catfish kind, and its sequence is as shown in SEQ ID NO:1 or as shown in SEQ ID NO:2.
For solving the problems of the technologies described above, four of technical scheme of the present invention is: a kind of primer detecting catfish kind, and its sequence is as shown in SEQ ID NO:3 or as shown in SEQ ID NO:4.
For solving the problems of the technologies described above, five of technical scheme of the present invention is: a kind of primer detecting catfish kind, and its sequence is as shown in SEQ ID NO:5 or as shown in SEQ ID NO:6.
For solving the problems of the technologies described above, six of technical scheme of the present invention is: a kind of primer detecting catfish kind, and its sequence is as shown in SEQ ID NO:7 or as shown in SEQ ID NO:8.
For solving the problems of the technologies described above, seven of technical scheme of the present invention is: a kind of primer detecting catfish kind, and its sequence is as shown in SEQ ID NO:9 or as shown in SEQ ID NO:10.
For solving the problems of the technologies described above, eight of technical scheme of the present invention is: a kind of primer detecting catfish kind, and its sequence is as shown in SEQ ID NO:11 or as shown in SEQ ID NO:12.
The preparation method of the primer of detection catfish kind of the present invention is this area customary preparation methods, is preferably synthetic complete sequence, or synthesizes primer of the present invention by business.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: the primer of the present invention's design is according to the COI gene for fish Mitochondrial DNA, and this gene existing specificity between the fish do not belonged to together, has conserved sequence again.Therefore, adopt the conserved sequence design PCR primer of this gene, ensure that the DNA that different fingerlings extracts has specificity, guarantee to be sequenced and to identify.The method differentiates that fingerling accuracy is high, and repeatable strong, error is little, and operator do not need deep species identification background to operate.
Meanwhile, 1) needed for this detection method, sample is a little block organization of fish, does not need complete fish body.2) employing has specific primer amplification cytochrome oxidase subunit I (COI) gene, guarantees accuracy and the uniqueness of authentication method.3) compare with traditional method, operator of the present invention do not need a large amount of working experiences, only need test according to operation steps.4) to have required detection time short for test kit of the present invention, and cost is low, and detected result specificity is high, the simple advantage of detected result decision method.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Embodiment 1 detects the Design and synthesis of catfish primer
1, according to NCBI (US National Biotechnology Information center) official website, search cytochrome oxidase subunit I (COI) gene of catfish, the COI gene obtaining Pacific herring (clupea pallasii) and oceanic herring (clupea harengus) amounts to 50 kinds, and the title of described COI gene fragment is as follows:
2, according to above-mentioned obtained gene, utilize DNAMAN5.0 software, by polygene sequence alignment, search the conservative region of these genes, the DNA sequence dna of the conservative region obtained is as shown in SEQ ID NO:13 in sequence table.
3, primer is prepared
According to cytochrome oxidase subunit I (COI) the gene conserved regions sequence obtained above, DNAMAN5.0 is adopted to design primer, design of primers optimum configurations is: product length 400 ~ 500bp, primer length 18 ~ 22bp, Tm value is 56 ~ 62 DEG C, and GC content is 45 ~ 55%.
Obtain initial primers right to 26, according to polygene sequence alignment result, reject the primer pair outside conservative region, simultaneously according to the result of pcr amplification reaction, reject the primer pair that cannot obtain specific band, 6 pairs of primers (Shanghai Major Biological Medical Technology Co., Ltd.) have been prepared in final selection, gained 6 pairs of primers called after primer 1 respectively, primer 2, primer 3, primer 4, primer 5 and primer 6.
12 nucleotide sequences of described 6 pairs of primers are respectively as shown in SEQ ID NO:1 ~ SEQ IDNO:12 in sequence table.
Embodiment 2 detects catfish kind
1, sample gene group DNA is extracted
Get commercially available catfish meat sample 1g, add liquid nitrogen and to mill 10min, get and pulverize the EP pipe that rear sample 0.1g joins 2ml, add cell pyrolysis liquid 1ml, Proteinase K 20 μ l, mixing;
At 62 DEG C of constant water bath box water-bath 20min, the centrifugal 5min of desk centrifuge 12000rpm, get supernatant liquor and carefully move in another centrifuge tube, add 2 times of volume isopropanol, can filament be seen after reversing mixing, choose with 100 μ l suction nozzles, dry;
Add 200 μ lTE and heavily wash dissolving, add equivalent Fen ︰ Lv Fang ︰ primary isoamyl alcohol (25 ︰ 24 ︰ 1) concussion mixing, the centrifugal 5min of 12000rpm;
Get upper solution to manage to another, add isopyknic Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) concussion mixing, the centrifugal 5min of 12000rpm;
Get upper solution to manage to another, add the 7.5mol/L ammonium acetate of 1/2 volume, then add the dehydrated alcohol of 2 times of volumes, after mixing, the centrifugal 10min of 12000rpm;
Carefully pour out supernatant liquor, tube wall residual droplets is removed by thieving paper, and with 1ml70% washing with alcohol throw out 3 times, the centrifugal 10min of 12000rpm, carefully outwells supernatant liquor, and EP pipe is put 37 DEG C, baking oven and dried 3h, obtains DNA about 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 1, the forward and reverse primer sequence in described primer 1 is respectively as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system is: gained genomic dna prepared by step 1 and add 200 μ lTE, again dissolve as template; Get DNA sample (template) 30 μ l, add 10 × damping fluid 10 μ l, dNTPMix8 μ l, Taq DNA Polymerase0.5 μ l, each 5 μ l of forward and reverse trip primer, supplement ddH
2o to 100 μ l.
C, pcr amplification program:
Pcr amplification program comprises the following steps: (1) 94 DEG C of denaturation 3min, (2) 94 DEG C of sex change 30s, (3) 58 DEG C of annealing 30s, (4) 72 DEG C extend 1min, wherein step (2) ~ (4) cycle index 30 times, (5) 72 DEG C extend 4min, (6) 4 DEG C of termination reactions.Obtain DNA solution 50 μ l, its concentration is about 100ng/ μ l.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample is through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), record gene order as shown in SEQ ID NO:14.
By the 5 '->3 ' sequence of gained goal gene sequencing result, Blast operating ratio pair is carried out in ncbi database, the sequence the highest with the sequence similarity of gained pcr amplification product is KC015290 (Maxident=100%), search sequence KC015290 in ncbi database, the kind information obtaining this gene order is: Clupea harengus, determines that this fish tissues sample belongs to oceanic herring fingerling (Clupeaharengus).
Embodiment 3 detects catfish kind
1, sample gene group DNA is extracted
Get catfish tissue sample, according to sample gene DNA group extracting method extracting tissue sample DNA described in embodiment 2.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 2, the forward and reverse primer sequence in described primer 2 is respectively as shown in SEQ ID NO:3 in sequence table and SEQ ID NO:4.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
Mg in b, PCR reaction system
2+concentration is 15mmol/L, dNTP concentration is 0.3mmol/L, and amplimer concentration is 0.3 μM, and Taq enzyme concentration is 0.1U/ μ L, and DNA profiling concentration is 100ng/ μ L.
C, pcr amplification program are (1) 93 DEG C of denaturation 2min, (2) 93 DEG C of sex change 28s, (3) 56 DEG C of annealing 28s, (4) 72 DEG C extend 1min, wherein step (2) ~ (4) cycle index 30 times, (5) 72 DEG C extend 3min, (6) 4 DEG C of termination reactions.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample is through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), record gene order as shown in SEQ ID NO:14.
By the 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, Blast runs, the sequence the highest with the sequence similarity of gained pcr amplification product is KC015290 (Maxident=99%), at ncbi database search sequence KC015290, obtaining its kind information is: Clupeaharengus, thus judges that this fish tissues sample belongs to oceanic herring fingerling (Clupea harengus).
Embodiment 4 detects catfish kind
1, sample gene group DNA is extracted
Get catfish tissue sample, according to sample gene group DNA extraction method extracting tissue sample DNA described in embodiment 2.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 3, the forward and reverse primer sequence in described primer 3 is respectively as shown in SEQ ID NO:5 in sequence table and SEQ ID NO:6.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
Mg in b, PCR reaction system
2+concentration is 10mmol/L, dNTP concentration is 0.2mmol/L, and amplimer concentration is 0.1 μM, and Taq enzyme concentration is 0.01U/ μ L, and DNA profiling concentration is 10ng/ μ L.
C, pcr amplification program are (1) 95 DEG C of denaturation 4min, (2) 95 DEG C of sex change 32s, (3) 60 DEG C of annealing 32s, (4) 72 DEG C extend 2min, wherein step (2) ~ (4) cycle index 35 times, (5) 72 DEG C extend 5min, (6) 4 DEG C of termination reactions.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample is through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), record gene order as shown in SEQ ID NO:15.
By the 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, run Blast and carry out sequence alignment, the sequence that the sequence the highest with the sequence similarity of gained pcr amplification product is is KF558314 (Max ident=99%), at ncbi database search sequence KF558314, obtaining its kind information is: Clupea pallasii, thus judges that this fish tissues sample belongs to clupea pallasi (Clupeapallasii).
Embodiment 5 detects catfish kind
1, sample gene group DNA is extracted
Get catfish tissue sample, according to sample gene group DNA extraction method extracting tissue sample DNA described in embodiment 2.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 4, the forward and reverse primer sequence in described primer 4 is respectively as shown in SEQ ID NO:7 in sequence table and SEQ ID NO:8.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system as described in Example 2.
C, pcr amplification program are as described in Example 2.
3, the order-checking of goal gene and comparison
Gained goal gene sample after amplification is delivered to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), records gene order as shown in SEQ ID NO:16 in sequence table.
By the 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, Blast runs, the sequence the highest with the sequence similarity of gained pcr amplification product is JF693642 (Max ident=99%), at NCBI search sequence JF693642, obtaining its kind information is: Clupea pallasii, thus judges that this fish tissues sample belongs to clupea pallasi (Clupea pallasii).
Embodiment 6 detects catfish kind
1, sample gene group DNA is extracted
Get catfish tissue sample, according to sample gene group DNA extraction method extracting tissue sample DNA described in embodiment 2.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 5, the forward and reverse primer sequence in described primer 5 is respectively as shown in SEQ ID NO:9 in sequence table and SEQ ID NO:10.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system as described in Example 2.
C, pcr amplification program are as described in Example 2.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample is through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), record gene order as shown in SEQ ID NO:17.
By the 5 '->3 ' sequence of gained goal gene sequencing result, Blast is run at ncbi database, the sequence the highest with the sequence similarity of gained pcr amplification product is KC015284 (Max ident=99%), at NCBI search sequence KC015284, obtaining its kind information is: Clupea harengus, thus judges that this fish tissues sample belongs to Atlantic Ocean catfish (Clupea harengus).
Embodiment 7 detects catfish kind
1, sample gene group DNA is extracted
Get catfish tissue sample, according to sample gene group DNA extraction method extracting tissue sample DNA described in embodiment 2.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 5, the forward and reverse primer sequence in described primer 5 is respectively as shown in SEQ ID NO:9 in sequence table and SEQ ID NO:10.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system as described in Example 2.
C, pcr amplification program are as described in Example 2.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample is through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), record gene order as shown in SEQ ID NO:18.
By the 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, Blast runs, the sequence the highest with the sequence similarity of gained pcr amplification product is: JF693642 (Max ident=99%), at NCBI search sequence JF693642, obtaining its kind information is: Clupea pallasii, thus judges that this fish tissues sample belongs to clupea pallasi (Clupea pallasii).
Comparative example 1
1, sample gene group DNA is extracted
Get commercially available grass carp structure of fish muscle, extracting method, with embodiment 2, obtains genomic dna about 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 1, the forward and reverse primer sequence in described primer 1 is respectively as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system as described in Example 2.
C, pcr amplification program as described in Example 2, the negative control in described pcr amplification program for being that template carries out pcr amplification with distilled water, positive controls wherein for the genomic dna of Atlantic Ocean catfish described in embodiment 2 for template carries out pcr amplification experiment.
3, amplification
Utilize PCR method described in embodiment 2, specific band cannot be amplified from the structure of fish muscle of negative control group and grass carp, can amplify specific band in positive controls, completely not containing the DNA profiling mated with primer 1 in interpret sample, therefore detected sample is not from catfish.
Comparative example 2
1, sample gene group DNA is extracted
Get commercially available crucian structure of fish muscle, the extracting method of genomic dna, with embodiment 2, obtains genomic dna about 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: Example 1 gained primer 2, the forward and reverse primer sequence in described primer 2 is respectively as shown in SEQ ID NO:3 in sequence table and SEQ ID NO:4.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system as described in Example 2.
C, pcr amplification program as described in Example 2, the negative control in this pcr amplification program for being that template carries out pcr amplification with distilled water, positive controls wherein for the genomic dna of Atlantic Ocean catfish described in embodiment 3 for template carries out pcr amplification experiment.
3, the order-checking of goal gene and comparison
Utilize PCR method described in embodiment 2, specific goal gene band cannot be amplified from the structure of fish muscle of negative control group and crucian, specific band can be amplified in positive controls, completely not containing the DNA profiling mated with primer 2 in interpret sample, therefore detected sample is not from catfish.
Comparative example 3
1, sample gene group DNA is extracted
Get the structure of fish muscle of different sources, the extracting method of its genomic dna, with embodiment 2, obtains genomic dna about 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: all primer 1-6 in Example 1.According to the concentration that synthetic primer is given, be mixed with the solution for standby that concentration is 10 μm of ol/L.
B, PCR reaction system as described in Example 2.
C, pcr amplification program as described in Example 2, the negative control in this pcr amplification program for being that template carries out pcr amplification with distilled water, positive controls wherein for the genomic dna of Atlantic Ocean catfish described in embodiment 3 for template carries out pcr amplification experiment.
3, the order-checking of goal gene and comparison
The analysis of goal gene, order-checking and comparison method are as described in Example 2.Cannot specific band be amplified in negative control group, can specific band be amplified in positive controls.
Acquired results is as shown in table 1-6:
The amplification of table 1 primer 1 and sequencing result
The amplification of table 2 primer 2 and sequencing result
The amplification of table 3 primer 3 and sequencing result
The amplification of table 4 primer 4 and sequencing result
The amplification of table 5 primer 5 and sequencing result
The amplification of table 6 primer 6 and sequencing result
As can be seen from the content shown in table 1-table 6, primer of the present invention both cannot amplify specific band from the nearly edge fish of clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus), also specific band in cannot increasing from the fish far away with the sibship of clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupeaharengus), therefore Auele Specific Primer of the present invention has genus specificity to clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus), the PCR primer that primer pair amplifies of the present invention obtains is checked order, just can detect the kind of testing sample.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. detect a method for clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus) kind, it is characterized in that, said method comprising the steps of:
(1) genomic dna of sample to be detected is extracted, with it for template, with the genomic dna of specificity amplification primer to amplification sample to be detected, the right sequence of described specificity amplification primer is respectively as SEQ ID NO:1 in sequence table and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ IDNO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8, or SEQ ID NO:9 and SEQ ID NO:10, or shown in SEQ ID NO:11 and SEQ ID NO:12
(2) step (1) gained pcr amplification product is carried out gel electrophoresis analysis, have the sample to be tested of specific band to be clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus).
2. the method for claim 1, it is characterized in that, described method also comprises step (3), step (1) gained pcr amplification product is checked order, the COI gene that sequencing result and catfish belong to is carried out sequence alignment, if COI gene matching degree >=99% of sample sequence and clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupeaharengus), then determine that sample to be tested is clupea pallasi (Clupeapallasii) or Atlantic Ocean catfish (Clupea harengus).
3. the method for claim 1, is characterized in that, in step (1), in described PCR reaction, the reaction system of PCR is: 1 × PCR reaction buffer, 10 ~ 15mmol/L Mg
2+, 0.2 ~ 0.3mmol/L dNTP, 0.1 ~ 0.3 μM of specificity amplification primer pair, Taq enzyme 0.01 ~ 0.1U/ μ L, DNA profiling 10 ~ 100ng/ μ L.
4. one kind is detected the test kit of clupea pallasi or oceanic herring fingerling class, it is characterized in that, it comprises specificity amplification primer pair, the right sequence of described specificity amplification primer is respectively as SEQ IDNO:1 in sequence table and SEQ ID NO:2, or SEQ ID NO:3 and SEQ ID NO:4, or SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8, or SEQ ID NO:9 and SEQ IDNO:10, or shown in SEQ ID NO:11 and SEQ ID NO:12.
5. detect a primer for clupea pallasi or oceanic herring fingerling class, it is characterized in that, its sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2.
6. detect a primer for clupea pallasi or oceanic herring fingerling class, it is characterized in that, its sequence is as shown in SEQ ID NO:3 and SEQ ID NO:4.
7. detect a primer for clupea pallasi or oceanic herring fingerling class, it is characterized in that, its sequence is as shown in SEQ ID NO:5 and SEQ ID NO:6.
8. detect a primer for clupea pallasi or oceanic herring fingerling class, it is characterized in that, its sequence is as shown in SEQ ID NO:7 and SEQ ID NO:8.
9. detect a primer for clupea pallasi or oceanic herring fingerling class, it is characterized in that, its sequence is as shown in SEQ ID NO:9 and SEQ ID NO:10.
10. detect a primer for clupea pallasi or oceanic herring fingerling class, it is characterized in that, its sequence is as shown in SEQ ID NO:11 and SEQ ID NO:12.
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| DNA条形码的应用进展及讨论;丁亮等;《环境昆虫学报》;20111231(第1期);全文 * |
| The Seafish Guid to DNA testing of seafood;anonymity;《seafish》;20131231;第3，8页 * |
| 李新光.基于DNA条形码的鱼片(肉)真伪鉴别技术研究.《中国优秀硕士学位论文全文数据库 工程科技I辑》.2014,(第5期),第49-56页. * |
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