CN103966347A - Method, kit and primers for detecting of species of Salmo - Google Patents
Method, kit and primers for detecting of species of Salmo Download PDFInfo
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Abstract
The invention discloses a method, kit and primers for detecting of species of Salmo. 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 primers, wherein the sequences of the specific amplification primers are respectively disclosed as SEQ ID NO:1-SEQ ID NO:10 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 Salmo salar or Oncorhynchus keta. 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, low cost, 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 salmon kind.
Background technology
Salmon is class migration fishes, is extensively distributed in the Atlantic Ocean, the Pacific Ocean, in freshwater lake, continent, also can find.Research is found, swims the salmon into streams, and 90% is all born in same streams.The salmon of Pacific Ocean kind, generally after breeding, several weeks just can be dead.Salmon, liver, spermary and head all have pharmaceutical use.Its meat have qi-restoratives labor, strengthening the spleen and stomach, warm stomach and in effect, can treat oedema, become thin, the disease such as maldigestion, swollen vexed swollen full, acid regurgitation, tic, swollen sore.Its liver can be obtained through refining Oils,glyceridic,cod-liver.Its spermary can be obtained through refining protamine and is mixed with multiple protamine preparation, adapts to the caused reaction of the excessive injecting heparin for the treatment of; It also has obvious anastalsis to some hemorrhage (as upper digestive tract acute hemorrhage, lung hemoptysis etc.), is to extract the important source material of making protamine injection liquid and insulinum protaminatum cumzinco.
Salmon is starting material source prepared by protamine.At present, for fish mirror method for distinguishing, be mainly to adopt traditional morphology differential method, complete fish body is carried out to morphological specificity comparison, complicated operation, strongly professional, still, its greatest drawback is the fish body that Structure of need is complete, can not have damage.If only take 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 salmon 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 salmon morphology differential method there is complicated operation, strongly professional defect.Adopt detection method of the present invention to detect the kind of salmon, 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: (1) extracts the genomic dna of sample to be detected, take 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 IDNO:7 and SEQ ID NO:8, or shown in SEQ ID NO:9 and SEQ ID NO:10
(2) step (1) gained pcr amplification product is carried out to gel electrophoresis analysis, have the sample to be tested of specific band to belong to atlantic salmon (Salmo salar) or Oncorhynchi (Oncorhynchus keta).
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, comprising: the 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 ℃ 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 ℃, also can, at 30-40 ℃ 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, with suction nozzle, choose, 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 ℃, baking oven and is dried 3-5 hour.
Wherein 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 ℃ of denaturation 2～4min, (2) 93～95 ℃ of sex change 28～32, (3) 56～60 ℃ of annealing 28～32s, (4) 72 ℃ are extended 1～2min, step (2)～(4) cycle index 30～35 times wherein, (5) 72 ℃ are extended 3～5min, (6) 4 ℃ of termination reactions.Be more preferably: (1) 94 ℃ of denaturation 3min, (2) 94 ℃ of sex change 30s, (3) 58 ℃ of annealing 30s, (4) 72 ℃ are extended 1min, step (2)～(4) cycle index 30 times wherein, (5) 72 ℃ are extended 4min, (6) 4 ℃ 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 salmon section is carried out to sequence alignment, matching degree >=99% of the COI gene of sample sequence and atlantic salmon (Salmo salar) or Oncorhynchi (Oncorhynchus keta), determines that sample to be tested is atlantic salmon (Salmo salar) or Oncorhynchi (Oncorhynchus keta).
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 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 of sequencing result and salmon genus carries out sequence alignment, if Ident value >=99% of sample sequence and atlantic salmon or Oncorhynchi COI gene determines that sample to be tested is atlantic salmon or Oncorhynchi.
Described order-checking and the sequence alignment method of step (3) is this area ordinary method, and the method for 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 salmon 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 shown in SEQ ID NO:9 and SEQ ID NO:10.
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 salmon 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 salmon 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 salmon 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 salmon 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 salmon kind, its sequence is as shown in SEQ ID NO:9 or as shown in SEQ ID NO:10.
The preparation method of the primer of detection salmon 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 being the COI gene of fish Mitochondrial DNA, and this gene is existing specificity between not equal fish, has again conserved sequence.Therefore, adopt the conserved sequence design PCR primer of this gene, guarantee that the DNA that increases out has the specificity of planting, and 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 to identify background.
Meanwhile, 1) the Yi 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 salmon primer with synthetic
1,, according to NCBI (U.S. state-run biotechnology information center) official website, the COI gene of searching three kinds of salmon Salmosalar, Oncorhynchus mykiss and Oncorhynchus keta amounts to 445 kinds, enumerates part as follows:
Cytochrome oxidase subunit I (COI) gene of salmon is as follows:
FJ999166 FJ998966 FJ998729
FJ999161 FJ998953 FJ998718
FJ999155 FJ998950 FJ998712
FJ999148 FJ998803 EU525057
FJ999131 FJ998799 HQ712702
FJ999122 FJ998788 HQ611128
FJ999118 FJ998777 GU440432
FJ999109 FJ998767 FJ918927
FJ998992 FJ998756 EF609449
FJ998986 FJ998745 FJ999539
FJ998973 FJ998739 FJ999511
FJ999508 JN007787 EU524353
FJ999503 JN007784 EU524349
FJ999485 JN007779 GU324184
FJ999472 HQ961026 HQ339988
FJ999469 HQ960962 EU752177
FJ999458 HQ025008
KC015282.1
KC015281.1
KC015280.1
GU324181.1
JF693779.1
JF693777.1
JF693778.1
JF693776.1
JF693775.1
JF693774.1
HQ611122.1
HQ611121.1
HQ611120.1
AM911176.1
2, according to resulting 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 IDNO:11 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 400～600bp, primer length 18～22bp, Tm value is 56～62 ℃, and GC content is 45～55%.
Obtain 22 pairs of initial primer pairs, according to polygene sequence alignment result, the primer pair of rejecting outside conservative region, according to the result of pcr amplification reaction, rejecting cannot obtain the primer pair of specific band simultaneously, and 5 pairs of primers (Shanghai Mei Ji biological medicine Science and Technology Ltd.) have been prepared in final selection, 5 pairs of primers of gained are called after primer 1 respectively, primer 2, primer 3, primer 4 and primer 5.
10 nucleotide sequences of described 5 pairs of primers are respectively as shown in SEQ ID NO:1～SEQ IDNO:10 in sequence table.
Embodiment 2 detects salmon kind
1, extract sample gene group DNA
Get commercially available salmon sample 1g, add the liquid nitrogen 10min that mills, get and pulverize the EP pipe that rear sample 0.1g joins 2ml, add cell pyrolysis liquid 1ml, Proteinase K 20 μ l, mix;
At 62 ℃ 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, with 100 μ l suction nozzles, chooses, and 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 ℃, 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, Tap 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 ℃ of denaturation 3min, (2) 94 ℃ of sex change 30s, (3) 58 ℃ of annealing 30s, (4) 72 ℃ are extended 1min, step (2)～(4) cycle index 30 times wherein, (5) 72 ℃ are extended 4min, (6) 4 ℃ 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 Mei Ji biological medicine Science and Technology Ltd.), records gene order as shown in SEQ ID NO:12.
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 JX960940 (Maxident=100%), search sequence JX960940 in ncbi database, the kind information that obtains this gene order is: Salmo salar, determines that this fish tissue sample belongs to atlantic salmon (Salmo salar).
Embodiment 3 detects salmon kind
1, extract sample gene group DNA
Get salmon 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 ℃ of denaturation 2min, (2) 93 ℃ of sex change 28s, (3) 56 ℃ of annealing 28s, (4) 72 ℃ are extended 1min, step (2)～(4) cycle index 30 times wherein, (5) 72 ℃ are extended 3min, (6) 4 ℃ 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 Mei Ji biological medicine Science and Technology Ltd.), records gene order as shown in SEQ ID NO:13.
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 KF597049 (Maxident=99%), at ncbi database search sequence KF597049, obtaining its kind information is: Salmosalar, thus judge that this fish tissue sample belongs to Atlantic salmon fingerling (Salmo salar).
Embodiment 4 detects salmon kind
1, extract sample gene group DNA
Get salmon 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 ℃ of denaturation 4min, (2) 95 ℃ of sex change 32s, (3) 60 ℃ of annealing 32s, (4) 72 ℃ are extended 2min, step (2)～(4) cycle index 35 times wherein, (5) 72 ℃ are extended 5min, (6) 4 ℃ 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 Mei Ji biological medicine Science and Technology Ltd.), records gene order as shown in SEQ ID NO:14.
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 FJ999537 (Max ident=99%), at ncbi database search sequence FJ999537, obtaining its kind information is: Salmo salar, thus judge that this fish tissue sample belongs to Atlantic salmon fingerling (Salmosalar).
Embodiment 5 detects salmon kind
1, extract sample gene group DNA
Get salmon tissue sample 1g, 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
After increasing, gained goal gene sample, through gel electrophoresis analysis, reclaims object fragment and delivers to order-checking company order-checking (Shanghai Mei Ji biological medicine Science and Technology Ltd.), records gene order as shown in SEQ ID NO:15.
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 HQ693265 (Maxident=99%), at NCBI search sequence HQ693265, obtaining its kind information is: Oncorhynchusketa, thus judge that this fish tissue sample belongs to Oncorhynchi (Oncorhynchus keta).
Embodiment 6 detects salmon kind
1, extract sample gene group DNA
Get salmon tissue sample 1g, 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 Mei Ji biological medicine Science and Technology Ltd.), records gene order as shown in SEQ ID NO:16.
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 JX960914 (Max ident=99%), at NCBI search sequence JX960914, obtaining its kind information is: Oncorhynchus keta, thus judge that this fish tissue sample belongs to Oncorhynchi (Oncorhynchus keta).
Comparative example 1
1, extract sample gene group DNA
Get commercially available 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 carries out pcr amplification for take distilled water as template for c, pcr amplification program, and positive controls is wherein carried out pcr amplification experiment for take the genomic dna of atlantic salmon described in embodiment 1 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 carp, amplify specific goal gene 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 salmon.
Comparative example 2
1, extract sample gene group DNA
Get the structure of fish muscle of goldfish, 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 carries out pcr amplification for take distilled water as template for c, pcr amplification program, and positive controls is wherein carried out pcr amplification experiment for take the genomic dna of atlantic salmon described in embodiment 2 as template.
3, pcr amplification result and analysis thereof
Utilize PCR method described in embodiment 2, cannot from the structure of fish muscle of negative control group and goldfish, 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 salmon.
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 1-5 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 carries out pcr amplification for take distilled water as template for c, pcr amplification program, and positive controls is wherein carried out pcr amplification experiment for take the genomic dna of atlantic salmon described in embodiment 2 as template.
3, pcr amplification result and analysis thereof
The analysis of goal gene, order-checking and comparison method 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 and analysis are as shown in table 1-5:
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
From the content shown in table 1-table 5, can find out, primer of the present invention both cannot amplify specific band from the nearly edge fish of atlantic salmon or Oncorhynchi, also cannot from atlantic salmon or Oncorhynchi sibship fish far away specific band in amplification, therefore Auele Specific Primer of the present invention has specificity to atlantic salmon or Oncorhynchi, the PCR product that primer amplification of the present invention is obtained checks order, and 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 (9)
1. a method that detects salmon kind, is characterized in that, said method comprising the steps of:
(1) extract the genomic dna of sample to be detected, take 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 shown in SEQ IDNO:9 and SEQ ID NO:10
(2) step (1) gained pcr amplification product is carried out to gel electrophoresis analysis, have the sample to be tested of specific band to belong to atlantic salmon (Salmo salar) or Oncorhynchi (Oncorhynchus keta).
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 salmon section is carried out to sequence alignment, matching degree >=99% of the COI gene of sample gene sequence and atlantic salmon (Salmo salar) or Oncorhynchi (Oncorhynchus keta), determines that sample to be tested is atlantic salmon (Salmo salar) or Oncorhynchi (Oncorhynchus keta).
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. a test kit that detects salmon 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 shown in SEQ ID NO:9 and SEQ ID NO:10.
5. a primer that detects salmon 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 salmon 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 salmon 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 salmon 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 salmon kind, is characterized in that, its sequence is as shown in SEQ ID NO:9 or as shown in SEQ ID NO:10.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371141A (en) * | 2018-11-29 | 2019-02-22 | 岛津企业管理（中国）有限公司 | Identify the method and specific primer pair of Atlantic salmon and rainbow trout |
| CN109536618A (en) * | 2018-12-13 | 2019-03-29 | 河南师范大学 | A kind of Jian Dings the methods of Minnow subfamily fish |
| CN111254202A (en) * | 2020-02-14 | 2020-06-09 | 南京工业大学 | Primer group, kit and detection method for detecting Atlantic salmon |
| CN112410438A (en) * | 2020-12-07 | 2021-02-26 | 中国农业科学院农业质量标准与检测技术研究所 | Primer and method for identifying source components of salmon, rainbow trout and salmon and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122386A (en) * | 2013-01-29 | 2013-05-29 | 中国水产科学研究院黄海水产研究所 | Molecular identification method for Atlantic salmon |
-
2014
- 2014-05-28 CN CN201410231413.0A patent/CN103966347A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122386A (en) * | 2013-01-29 | 2013-05-29 | 中国水产科学研究院黄海水产研究所 | Molecular identification method for Atlantic salmon |
Non-Patent Citations (1)
| Title |
|---|
| 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》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109371141A (en) * | 2018-11-29 | 2019-02-22 | 岛津企业管理（中国）有限公司 | Identify the method and specific primer pair of Atlantic salmon and rainbow trout |
| CN109536618A (en) * | 2018-12-13 | 2019-03-29 | 河南师范大学 | A kind of Jian Dings the methods of Minnow subfamily fish |
| CN111254202A (en) * | 2020-02-14 | 2020-06-09 | 南京工业大学 | Primer group, kit and detection method for detecting Atlantic salmon |
| CN112410438A (en) * | 2020-12-07 | 2021-02-26 | 中国农业科学院农业质量标准与检测技术研究所 | Primer and method for identifying source components of salmon, rainbow trout and salmon and application |
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Application publication date: 20140806 |