CN103966346A - Method, kit and primers for detecting of species of Clupea - Google Patents
Method, kit and primers for detecting of species of Clupea Download PDFInfo
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Abstract
The invention discloses a method, kit and primers for detecting of species of Clupea. The method comprises the following steps: (1) by using genome DNA (deoxyribonucleic acid) of a sample to be detected as a template, amplifying the genome DNA of the sample to be detected by using specific amplification primer pairs, wherein the sequences of the specific amplification primer pairs are respectively disclosed as SEQ ID NO:1-SEQ ID NO:12 in the sequence table; and (2) carrying out gel electrophoresis analysis on the PCR (polymerase chain reaction) amplification product, wherein the sample to be detected with specific strips belongs to Clupea pallasii or Clupea harengus. Compared with the traditional method, the method disclosed by the invention is accurate and unique. The operating personnel does not need much working experience, and only needs to do the experiment according to the operation steps. The kit has the advantages of short required detection time, high specificity of detection results and simple detection result judgment method.
Description
Technical field
The present invention relates to fish detection field, be specifically related to a kind of method and detection kit and primer that detects catfish kind.
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 to the U.S.West Coast waters of Alaska Bering Sea produces clupea pallasi (Clupea pallasi).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, and deoxynucleotide and arginine are to extract the important source material of making protamine injection liquid and insulinum protaminatum cumzinco.
Catfish is starting material source prepared by protamine.At present, be mainly to adopt traditional morphology differential method for fish mirror method for distinguishing, complete fish body is carried out to morphological specificity comparison, but this authentication method greatest drawback is the fish body that Structure of need is complete, can not there is damage, and complicated operation, strongly professional.If only taking fish tissue sample as material, adopt traditional entirety morphology means, cannot carry out fish discriminating.
Summary of the invention
The technical problem to be solved in the present invention is to overcome traditional catfish traditional form differential method needs complete fish body carry out morphological specificity comparison, the fish body that Structure of need is complete, can not have damage, make traditional catfish morphology differential method there is 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 that detects catfish kind, said method comprising the steps of: (1) genomic dna of sample to be detected increases, 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 to gel electrophoresis analysis, the sample to be tested that has specific band is clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus).
According to the present invention, described in step (1), sample to be tested is the sample to be tested of this area routine, is preferably a part for fish body to be measured, the preferably flesh of fish, internal organ, fish head, the fish sperm, one or more in roe and fish scale.
The method of 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 tissue sample and add the liquid nitrogen 10min that mills, or 100 DEG C dried 5-8 hour, 10min mills after to be dried.
The sample of getting after pulverizing adds EP pipe, adds cell pyrolysis liquid 1ml, adds Proteinase K 20 μ l, mixes.
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, intermittently shake EP pipe 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 Virahols, after reversing mixes, can see filament, choose with suction nozzle, dry.
Add 200 μ lTE heavily to wash dissolving, add the phenol of equivalent: chloroform: primary isoamyl alcohol (25:24:1) concussion mixes, the centrifugal 5-10min of rotating speed 12000-13000rpm.
Get upper solution and manage to another, add isopyknic chloroform: primary isoamyl alcohol (24:1), concussion mixes, the centrifugal 5-10min of rotating speed 12000-13000rpm.
Get upper solution and 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, thieving paper is removed tube wall residual droplets.
With the washing with alcohol throw out of 1ml70% 2-3 time, 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 of the reaction of PCR described in step (1) is preferably 1 × PCR reaction buffer, 10～15mmol/L Mg
2+, 0.2～0.3mmol/L dNTP, 0.1～0.3 μ M 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 μ M specificity amplification primer pair, 0.04U/ μ LTaq enzyme and 50ng/ μ L DNA profiling.
The amplification program of the reaction of PCR described in step (1) 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 are extended 1～2min, wherein step (2)～(4) cycle index 30～35 times, (5) 72 DEG C are extended 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 are extended 1min, wherein step (2)～(4) cycle index 30 times, (5) 72 DEG C are extended 4min, (6) 4 DEG C of termination reactions.
Method of the present invention more preferably also comprises step (3): step (1) gained pcr amplification product is checked order, the COI gene of sequencing result and catfish genus is carried out to 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 by Ident value representation, and Ident value refers to the matching degree of two sections of nucleotide sequences, and this value more approaches 100%, illustrates that the similarity of two nucleotide sequences is higher.Consider the accuracy impact of gene amplification and order-checking, in the time of Ident value >=99%, just can judge that the gene of species to be measured and goal gene are from same species.Therefore in the time that the COI gene of sequencing result and catfish genus carries out sequence alignment, if Ident value >=99% of sample sequence and clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus) COI gene, determines that sample to be tested is clupea pallasi or Atlantic Ocean catfish.
Described order-checking and the sequence alignment method of step (3) is 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 that detects 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 that detects catfish kind, 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 that detects catfish kind, 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 that detects catfish kind, 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 that detects catfish kind, 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 that detects catfish kind, 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 that detects catfish kind, 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 the conventional preparation method in this area, is preferably synthetic complete sequence, or synthesizes primer of the present invention by business.
Meeting on the basis of 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 be commercially available obtaining all.
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 that do not belong to together, has again conserved sequence.Therefore, adopt the conserved sequence design PCR primer of this gene, ensure that the DNA that different fingerlings extracts has specificity, guarantees 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 qualification background to operate.
Meanwhile, 1) the little block organization that the required sample of this detection method is fish, does not need complete fish body.2) adopt and there is specific primer amplification cytochrome oxidase subunit I (COI) gene, guarantee 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) it is short that test kit of the present invention has required detection time, 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, according to ordinary method and condition, or selects according to catalogue.
Embodiment 1 detects the design of catfish primer with synthetic
1, according to NCBI (state-run biotechnology information center of the U.S.) official website, search cytochrome oxidase subunit I (COI) gene of catfish, the COI gene that obtains 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 obtaining is as shown in SEQ ID NO:13 in sequence table.
3, prepare primer
According to cytochrome oxidase subunit I (COI) the gene conservative region sequence obtaining above, adopt DNAMAN5.0 design primer, design of primers parameter is set to: product length 400～500bp, primer length 18～22bp, Tm value is 56～62 DEG C, and GC content is 45～55%.
Obtain initial primer pair 26 right, according to polygene sequence alignment result, reject the primer pair outside conservative region, simultaneously according to the result of pcr amplification reaction, rejecting cannot obtain the primer pair of specific band, 6 pairs of primers (Shanghai Major Biological Medical Technology Co., Ltd.) have been prepared in final selection, gained 6 is to primer 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, extract sample gene group DNA
Get commercially available catfish meat sample 1g, add the liquid nitrogen 10min that mills, get the EP pipe that pulverizes rear sample 0.1g and join 2ml, add cell pyrolysis liquid 1ml, Proteinase K 20 μ l, mix;
At 62 DEG C of constant water bath box water-bath 20min, the centrifugal 5min of desk centrifuge 12000rpm, gets supernatant liquor and carefully moves in another centrifuge tube, adds 2 times of volume Virahols, after reversing mixes, can see filament, chooses with 100 μ l suction nozzles, dries;
Add 200 μ lTE heavily to wash dissolving, add equivalent Fen ︰ Lv Fang ︰ primary isoamyl alcohol (25 ︰ 24 ︰ 1) concussion to mix, the centrifugal 5min of 12000rpm;
Get upper solution and manage to another, add isopyknic Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) concussion to mix, the centrifugal 5min of 12000rpm;
Get upper solution and 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, thieving paper is removed tube wall residual droplets, uses 1ml70% washing with alcohol throw out 3 times, and the centrifugal 10min of 12000rpm, carefully outwells supernatant liquor, and EP pipe is put 37 DEG C, baking oven and dried 3h, obtains DNA approximately 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are: step 1 is prepared to gained genomic dna 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, the 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 are extended 1min, wherein step (2)～(4) cycle index 30 times, (5) 72 DEG C are extended 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, through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), records gene order as shown in SEQ ID NO:14.
By 5 '->3 ' sequence of gained goal gene sequencing result, in ncbi database, carry out Blast operating ratio pair, the sequence the highest with the sequence similarity degree of gained pcr amplification product is KC015290 (Maxident=100%), search sequence KC015290 in ncbi database, the kind information that obtains this gene order is: Clupea harengus, determines that this fish tissue sample belongs to oceanic herring fingerling (Clupeaharengus).
Embodiment 3 detects catfish kind
1, extract sample gene group DNA
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: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
Mg in b, PCR reaction system
2+concentration is 15mmol/L, and dNTP concentration is 0.3mmol/L, and amplimer concentration is 0.3 μ M, and Taq enzyme concn 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 are extended 1min, wherein step (2)～(4) cycle index 30 times, (5) 72 DEG C are extended 3min, (6) 4 DEG C of termination reactions.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample, through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), records gene order as shown in SEQ ID NO:14.
By 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, Blast operation, the sequence the highest with the sequence similarity degree of gained pcr amplification product is KC015290 (Maxident=99%), at ncbi database search sequence KC015290, obtaining its kind information is: Clupeaharengus, thus judge that this fish tissue sample belongs to oceanic herring fingerling (Clupea harengus).
Embodiment 4 detects catfish kind
1, extract sample gene group DNA
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: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
Mg in b, PCR reaction system
2+concentration is 10mmol/L, and dNTP concentration is 0.2mmol/L, and amplimer concentration is 0.1 μ M, and Taq enzyme concn 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 are extended 2min, wherein step (2)～(4) cycle index 35 times, (5) 72 DEG C are extended 5min, (6) 4 DEG C of termination reactions.
3, the order-checking of goal gene and comparison
After increasing, gained goal gene sample, through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), records gene order as shown in SEQ ID NO:15.
By 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, operation Blast carries out sequence alignment, the sequence that is with the highest sequence of the sequence similarity degree of gained pcr amplification product is KF558314 (Max ident=99%), at ncbi database search sequence KF558314, obtaining its kind information is: Clupea pallasii, thus judge that this fish tissue sample belongs to clupea pallasi (Clupeapallasii).
Embodiment 5 detects catfish kind
1, extract sample gene group DNA
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: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are 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.), record gene order as shown in SEQ ID NO:16 in sequence table.
By 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, Blast operation, the sequence the highest with the sequence similarity degree of gained pcr amplification product is JF693642 (Max ident=99%), at NCBI search sequence JF693642, obtaining its kind information is: Clupea pallasii, thus judge that this fish tissue sample belongs to clupea pallasi (Clupea pallasii).
Embodiment 6 detects catfish kind
1, extract sample gene group DNA
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: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are 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, through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), records gene order as shown in SEQ ID NO:17.
By 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database operation Blast, the sequence the highest with the sequence similarity degree of gained pcr amplification product is KC015284 (Max ident=99%), at NCBI search sequence KC015284, obtaining its kind information is: Clupea harengus, thus judge that this fish tissue sample belongs to Atlantic Ocean catfish (Clupea harengus).
Embodiment 7 detects catfish kind
1, extract sample gene group DNA
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: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are 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, through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Major Biological Medical Technology Co., Ltd.), records gene order as shown in SEQ ID NO:18.
By 5 '->3 ' sequence of gained goal gene sequencing result, at ncbi database, Blast operation, the sequence the highest with the sequence similarity degree of gained pcr amplification product is: JF693642 (Max ident=99%), at NCBI search sequence JF693642, obtaining its kind information is: Clupea pallasii, thus judge that this fish tissue sample belongs to clupea pallasi (Clupea pallasii).
Comparative example 1
1, extract sample gene group DNA
Get commercially available grass carp structure of fish muscle, extracting method, with embodiment 2, obtains genomic dna approximately 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are as described in Example 2.
As described in Example 2, the negative control in described pcr amplification program is for carry out pcr amplification taking distilled water as template for c, pcr amplification program, and positive controls is wherein to carry out pcr amplification experiment taking the genomic dna of Atlantic Ocean catfish described in embodiment 2 as template.
3, amplification
Utilize PCR method described in embodiment 2, cannot from the structure of fish muscle of negative control group and grass carp, amplify specific band, in positive controls, can amplify specific band, in interpret sample, completely not containing the DNA profiling mating with primer 1, therefore the sample that detects is not from catfish.
Comparative example 2
1, extract sample gene group DNA
Get commercially available crucian structure of fish muscle, the extracting method of genomic dna, with embodiment 2, obtains genomic dna approximately 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: get embodiment 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 given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are as described in Example 2.
As described in Example 2, the negative control in this pcr amplification program is for carry out pcr amplification taking distilled water as template for c, pcr amplification program, and positive controls is wherein to carry out pcr amplification experiment taking the genomic dna of Atlantic Ocean catfish described in embodiment 3 as template.
3, the order-checking of goal gene and comparison
Utilize PCR method described in embodiment 2, cannot from the structure of fish muscle of negative control group and crucian, amplify specific goal gene band, in positive controls, can amplify specific band, in interpret sample, completely not containing the DNA profiling mating with primer 2, therefore the sample that detects is not from catfish.
Comparative example 3
1, extract sample gene group DNA
Get the structure of fish muscle of different sources, the extracting method of its genomic dna, with embodiment 2, obtains genomic dna approximately 0.2 μ g.
2, PCR method amplifying target genes
(1) PCR reaction
A, primer: get all primer 1-6 in embodiment 1.According to the given concentration of synthetic primer, be mixed with the solution for standby that concentration is 10 μ mol/L.
B, PCR reaction system are as described in Example 2.
As described in Example 2, the negative control in this pcr amplification program is for carry out pcr amplification taking distilled water as template for c, pcr amplification program, and positive controls is wherein to carry out pcr amplification experiment taking the genomic dna of Atlantic Ocean catfish described in embodiment 3 as template.
3, the order-checking of goal gene and comparison
Analysis, order-checking and the comparison method of goal gene are as described in Example 2.In negative control group, specific band cannot be amplified, in positive controls, specific band can be amplified.
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
From the content shown in table 1-table 6, can find out, 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 cannot from the sibship of clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupeaharengus) fish far away amplification in specific band, therefore Auele Specific Primer of the present invention has genus specificity to clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus), the primer pair of the present invention PCR product obtaining that increases is checked order, just can detect the kind of testing sample.
Should be understood that, after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a method that detects catfish kind, is characterized in that, said method comprising the steps of:
(1) extract the genomic dna of sample to be detected, taking it as template, genomic dna with specificity amplification primer to the sample to be detected that increases, 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 ID NO:5 and SEQ ID NO:6, or SEQ ID NO:7 and SEQ ID NO:8, or SEQ IDNO: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 to gel electrophoresis analysis, the sample to be tested that has specific band is clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupeaharengus).
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 of sequencing result and catfish genus is carried out to sequence alignment, if COI gene matching degree >=99% of sample sequence and clupea pallasi (Clupea pallasii) or Atlantic Ocean catfish (Clupea harengus), determines that sample to be tested is clupea pallasi (Clupea pallasii) 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 μ M 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 catfish kind, it is characterized in that, 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 IDNO: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 ID NO:10, or shown in SEQID NO:11 and SEQ ID NO:12.
5. a primer that detects catfish kind, is characterized in that, its sequence is as shown in SEQ ID NO:1 or as shown in SEQ ID NO:2.
6. a primer that detects catfish kind, is characterized in that, its sequence is as shown in SEQ ID NO:3 or as shown in SEQ ID NO:4.
7. a primer that detects catfish kind, is characterized in that, its sequence is as shown in SEQ ID NO:5 or as shown in SEQ ID NO:6.
8. a primer that detects catfish kind, is characterized in that, its sequence is as shown in SEQ ID NO:7 or as shown in SEQ ID NO:8.
9. a primer that detects catfish kind, is characterized in that, its sequence is as shown in SEQ ID NO:9 or as shown in SEQ ID NO:10.
10. a primer that detects catfish kind, is characterized in that, its sequence is as shown in SEQ ID NO:11 or as shown in SEQ ID NO:12.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104988228A (en) * | 2015-07-09 | 2015-10-21 | 国家海洋局第三海洋研究所 | Primer group and method for detecting fish CO I genes through nested PCR method |
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| ANONYMITY: "The Seafish Guid to DNA testing of seafood", 《SEAFISH》, 31 December 2013 (2013-12-31), pages 3 - 8 * |
| 丁亮等: "DNA条形码的应用进展及讨论", 《环境昆虫学报》, no. 1, 31 December 2011 (2011-12-31) * |
| 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》, no. 5, 15 May 2014 (2014-05-15), pages 49 - 56 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104988228A (en) * | 2015-07-09 | 2015-10-21 | 国家海洋局第三海洋研究所 | Primer group and method for detecting fish CO I genes through nested PCR method |
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