CN106636364A - Multi-PCR kit for rapidly identifying Dragonfish type and identifying method thereof - Google Patents
Multi-PCR kit for rapidly identifying Dragonfish type and identifying method thereof Download PDFInfo
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- CN106636364A CN106636364A CN201611043139.XA CN201611043139A CN106636364A CN 106636364 A CN106636364 A CN 106636364A CN 201611043139 A CN201611043139 A CN 201611043139A CN 106636364 A CN106636364 A CN 106636364A
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Abstract
The invention provides a multi-PCR kit for rapidly identifying Dragonfish type and an identifying method thereof; the kit includes 1, three upstream primers and three downstream primers; 2, positive comparison product; 3, PCR reaction fluid; meanwhile, the method also provides a method for rapidly identifying the Dragonfish type, wherein the method includes steps of 1), selecting three upstream primers and three downstream primers; 2), preparing a gene group DNA template; 3), respectively adding the upstream primers and the downstream primers in the gene group DNA template prepared by the positive controlled DNA template and the Dragonfish to be tested, so as to perform PCR amplified reaction;4). Taking 5 miu L products after amplification and applying samples to 2.5% of sepharose gel; taking 50 bp Marker (stripes 50, 100, 150, 200, 250, 300, 350, 400, 450, 500) as standard molecule reference and performing electrophoresis; applying the electrophoresis result to the analysis of an ultraviolet gel imaging system; 5), judging result. The kit and the method can improve the identifying efficiency, save time, and save the identifying cost.
Description
Technical field
The invention belongs to molecular biology authentication technique field, and in particular to a kind of PCR examinations of quick discriminating dragonfish species
Agent box and its discrimination method.
Background technology
The natural, graceful and poised prolate of dragonfish build, mouth is wealthy must be long, and trunk is covered with marshalling, the very large scale of radiance flicker, trip
The sedate prestige of imposing manner, is greatly pursued by pet fish fan when dynamic, is to view and admire value highest fancy fishes on fish market,
Especially in Chinese area, some dragonfish prices exceed million yuan.
Asia dragonfish (Scleropages formosus), Margarita dragonfish (Scleropagesjardinii) and silver-colored dragonfish
(Osteoglossum bicirrhosum) is current dragonfish most popular in the world.Asia dragonfish and Margarita dragonfish belong to os osseum
Tongue fish belongs to, and silver-colored dragonfish belongs to osteoglossid category.Due to the adult fish form and ornamental value most diverse of these dragonfish, its economic valency
Value also has greatest differences, but the germling form of these dragonfish is quite similar, it is difficult to differentiated by morphological method.Therefore,
In the urgent need to setting up a kind of fast and accurately molecular biological variety identification method.Fish mitochondrial gene has evolutionary rate fast, thing
Interspecific difference is larger and thing intraspecies variation very little, and copy number is more, and each cell contains mitochondrial DNA 100～10 000 to be copied
Shellfish.Therefore, just with differentiator kind, and species discriminating can be widely used in using shorter mitochondrial gene fragment.This patent pin
The Mitochondrial gene sequence of above-mentioned three kinds of dragonfish, designs specific primer, can well differentiate three kinds of dragonfish.
The content of the invention
The scope of the present invention does not receive this section content of the invention only by appended claims defined, in any degree
Statement limited.
It is an object of the invention to provide the multiple PCR reagent kit and its method of a kind of quick discriminating dragonfish species；This
The bright a kind of method for being quick, accurate, sensitive multiplex PCR detection and identifying Asia dragonfish, silver-colored dragonfish and Margarita dragonfish, be point
Sub- biological method differentiates that Asia dragonfish, silver-colored dragonfish and Margarita dragonfish provide selection.Identification efficiency is not only improved, the time is saved,
And saving differentiates cost.
In order to reach above-mentioned purpose, the solution of the present invention is：
A kind of multiple PCR reagent kit of quick discriminating dragonfish species, including：1st, 3 forward primer and 3 downstream primers；
2nd, positive reference substance；3rd, PCR reactant liquors；
A kind of multiplex PCR discrimination method of quick discriminating dragonfish species is：
1) 3 forward primer and 3 downstream primers are selected；
2) take positive reference substance and sample to be checked prepares respectively genomic DNA template；
3) respectively positive reference substance DNA profiling and dragonfish to be checked prepare genomic DNA template in add on described
Trip primer and downstream primer carry out pcr amplification reaction；
4) the μ L point samples of product 5 after amplification are taken in 2.5% agarose gel, wherein containing 0.1～0.5 μ g/mL ethidium bromides,
, as standard molecule reference, carried out with 50bp Marker (band 50,100,150,200,250,300,350,400,450,500)
Electrophoresis；Electrophoresis result is analyzed with ultraviolet gel imaging system；
5) measuring samples are compared by result judgement with positive reference substance analysis result, measuring samples and positive control
Simultaneously 468bp bands are amplified, be judged to Asia dragonfish；Measuring samples amplify 325bp bands simultaneously with positive control,
It is judged to silver-colored dragonfish；Measuring samples amplify 283bp bands simultaneously with positive control, are judged to Margarita dragonfish.
Described 3 forward primer and the primer sequence of 3 downstream primers are respectively：
Forward primer SF-For：5-CCTATGTACTCACCACCCTAATC-3；
Downstream primer SF-Rev：5'-CTCCTAGGGTAGTGAGAAGGGC-3'；
Forward primer OB-For：5'-CATATTTCTTAAGGTACTCTG-3'；
Downstream primer OB-Rev：5'-GAATAGCACACCGGTGTAGGAT-3'；
Forward primer SJ-For：5'GCCTGCTACTGCAGTTGTAGTT 3'；
Downstream primer SJ-Rev-3：5'-GAAAGTCAGGATTATGAAGAT-3'.
Described PCR reaction conditions are as follows：
95 DEG C of 3min of denaturation
95 DEG C of 30s of degeneration
58 DEG C of 30s of annealing
Extend 72 DEG C of 30s, carry out 30 cyclic amplifications；
Extend 72 DEG C of extension 5min eventually.
The composition of the PCR reactant liquors is as follows：
| Composition | Content |
| 10 × amplification buffer | 5μl |
| 4 kinds of dNTP mixture | Each 200 μm of ol/L |
| Primer | Each 0.5 μm of ol/L |
| DNA profiling | 2μg |
| Taq archaeal dna polymerases | 2ug |
| MgCl2 | 1.5mmol/L |
| ddH2O | 45μl |
Wherein described amplification buffer is 100mMTris-HCl (8.8,25 DEG C of pH), 500mM KCl, 0.8% (v/v)
Nonidet P40
4 kinds of wherein described dNTP mixture refer to：Triphosphoric acid adenyl-deoxyribonucleotide dATP, deoxythymidine triphosphate
DTTP, deoxyguanosine triphosphate trisodium dGTP, deoxycytidine triphosphate dCTP.(BBI)
Wherein described positive reference substance is：The mitochondrial DNA of 3 kinds of dragonfish (Asia dragonfish, silver-colored dragonfish and Margarita dragonfish)
Plasmid standard.
Method prepared by described dragonfish DNA profiling to be checked is：
A. dragonfish tissue is taken, adds 400 μ l lysates to mix, be placed in 37 DEG C of water-bath 1h；
B. the sodium chloride solution of 200 μ l 5mol/L is subsequently adding, is centrifuged after mixing, centrifugal condition 13000rpm,
15min；
C. supernatant is taken, with phenol extraction 2 times, chloroform 1 time；
D. plus two volumes dehydrated alcohol, 1/10 volume, the potassium acetate of 3mol/L, pH8.0, -20 DEG C of preservation 1h；
E. above-mentioned solution is centrifuged, centrifugal condition 13000rpm, 15min, abandons supernatant, 70% ethanol of precipitate
Wash 2 times；
F. it is placed in after drying at room temperature, in being dissolved in 50 μ lTE solution (Shanghai Suo Laibao bio tech ltd), puts 4 DEG C of guarantors
Deposit standby.
Described cracking formula of liquid is：40mmol/L Tris- acetic acid, 20mmol/L sodium acetates, 1mmol/L EDTA, 1%
Sodium lauryl sulphate (SDS), pH7.8.
Beneficial effects of the present invention are：The present invention establishes the multiple PCR method inspection using the competitive annealing of a plurality of primer
The method surveyed and differentiate 3 kinds of dragonfish, and the detection test of Jing actual samples, do not find false positive results, show that the method conscientiously may be used
OK.This method adopts PCR amplification techniques so that the sensitivity of 3 kinds of dragonfish of detection is greatly improved.Due to PCR of this method Jing
3 kinds of dragonfish can be differentiated, it is not necessary to confirmation identification is carried out respectively to every kind of dragonfish, experimental period is saved, accelerate detection speed
Degree.In sum：The dragonfish detection of the present invention and authentication method have the advantages that sensitivity height, high specificity, easy to operate, compared with
Traditional Morphological Identification method has higher repeatability, more quick compared with regular-PCR, easy, is capable of achieving to 3 kinds of dragonfish
Quick, accurate, special detection and analysis.
Description of the drawings
Fig. 1 is the multiplexed PCR amplification product electrophoretogram of plasmid standard positive control of the present invention and dragonfish sample, wherein：
1：Silver-colored dragonfish sample (OB)；2：Margarita dragonfish sample (SJ)；3：Asia dragonfish sample (SF)；4：The silver-colored dragonfish positive is right
According to；5：Margarita dragonfish positive control；6：Asia dragonfish positive control；M：50bp DNA Ladder Marker.
Specific embodiment
The present invention is described in further detail below in conjunction with drawings and Examples, does not therefore limit the present invention to institute
In the scope of embodiments stated.
Embodiment 1
Materials and methods
1. material
The Asia dragonfish that 1.1 raisers provide, silver-colored dragonfish and Margarita dragonfish sample；
1.2 key instrument reagents
2. method
2.1 mitochondrial DNAs are extracted
1. extract mitochondrial DNA from tissue, weigh 100mg samples, add 400 μ l lysates to be homogenized to invisible
Obvious piece of tissue；
2. 37 DEG C of water-bath 1h are placed in；
3. the sodium chloride solution of 200 μ l 5mol/L is added, is mixed and 15min is centrifuged after 13000rpm；
4. supernatant is taken, with phenol extraction 2 times, chloroform 1 time；
5. plus two volumes dehydrated alcohol, 1/10 volume, the potassium acetate of 3mol/L, pH8.0, -20 DEG C of preservation 1h,
6.13000rpm is centrifuged 15min, abandons supernatant, and precipitate washes 2 times with 70% ethanol；7. it is placed in after drying at room temperature,
In being dissolved in 50 μ l TE solution (Shanghai Suo Laibao bio tech ltd), put 4 DEG C and save backup.
2.2 PCR react
PCR amplification system：
PCR reaction conditions：
| Cycle | Cycle Point |
| 95 DEG C of Hold@, 3min | |
| Cycling(30repeats) | 95 DEG C of 1@of Step, hold 30s |
| 58 DEG C of 2@of Step, hold 30s | |
| 72 DEG C of 3@of Step, hold 30s | |
| 72 DEG C of Hold@, 5min |
2.3 2.5% agarose gel electrophoresiies are detected.
The μ L point samples of product 5 after amplification are taken in 2.5% agarose gel (containing 0.5 μ g/mL ethidium bromides), with 50bp
Marker is standard molecule reference, carries out electrophoresis.Voltage 100v run 10 minutes, then 50v run two hours.Electrophoresis result is purple
Outer gel imaging system analysis.
3rd, experimental result
Annealing temperature is set to 55 DEG C, and special primer is expanded, knowable to electrophoresis detection result：1：Silver-colored dragonfish sample
(OB)；2：Margarita dragonfish sample (SJ)；3：Asia dragonfish sample (SF)；4：Silver-colored dragonfish positive control；5：The Margarita dragonfish positive is right
According to；6：Asia dragonfish positive control；M：50bp Marker.
Claims (9)
1. a kind of multiple PCR reagent kit of quick discriminating dragonfish species, including：(1), 3 forward primer and 3 downstream primers；
(2), positive reference substance；(3), PCR reactant liquors；Wherein, described 3 forward primer and the sequence of 3 downstream primers are respectively：
Forward primer SF-For：5-CCTATGTACTCACCACCCTAATC-3；
Downstream primer SF-Rev：5'-CTCCTAGGGTAGTGAGAAGGGC-3'；
Forward primer OB-For：5'-CATATTTCTTAAGGTACTCTG-3'；
Downstream primer OB-Rev：5'-GAATAGCACACCGGTGTAGGAT-3'；
Forward primer SJ-For：5'GCCTGCTACTGCAGTTGTAGTT 3'；
Downstream primer SJ-Rev-3：5'-GAAAGTCAGGATTATGAAGAT-3'.
2. a kind of discrimination method of quick discriminating dragonfish species is：
1) 3 forward primer and 3 downstream primers are selected；
2) take positive reference substance and dragonfish sample to be checked prepares respectively genomic DNA template；
3) respectively positive control DNA profiling and dragonfish to be checked prepare genomic DNA template in add the forward primer
Pcr amplification reaction is carried out with downstream primer；
4) the μ L point samples of product 5 after amplification are taken in 2.5% agarose gel, wherein containing 0.1～0.5 μ g/mL ethidium bromides, with
50bp Marker are standard molecule reference, carry out electrophoresis；Electrophoresis result is analyzed with ultraviolet gel imaging system；
5) result judgement, measuring samples are compared with positive control electrophoresis result, determine dragonfish species.
3. the multiple PCR reagent kit of a kind of quick discriminating dragonfish species according to claim 1, it is characterised in that wherein
The composition of described PCR reactant liquors is：
4. the discrimination method of a kind of quick discriminating dragonfish species according to claim 2, it is characterised in that wherein described
Pcr amplification reaction is carried out as follows：
5. according to claim 3, wherein the amplification buffer is 100mMTris-HCl (8.8,25 DEG C of pH), 500mM KCl,
0.8% (v/v) Nonidet P40.
6. according to claim 3, wherein 4 kinds of described dNTP mixture refer to：Triphosphoric acid adenyl-deoxyribonucleotide dATP, deoxidation
Thymidine triphosphate dTTP, deoxyguanosine triphosphate trisodium dGTP, deoxycytidine triphosphate dCTP.
7. the multiple PCR reagent kit of a kind of quick discriminating dragonfish species according to claim 1, it is characterised in that wherein
Described positive reference substance is：The Mitochondrial plasmid DNA standard substance of 3 kinds of dragonfish (Asia dragonfish, silver-colored dragonfish and Margarita dragonfish).
8. the discrimination method of a kind of quick discriminating dragonfish species according to claim 2, it is characterised in that wherein described
Method prepared by dragonfish DNA profiling to be checked is：
A. dragonfish tissue is taken, adds 400 μ l lysates to mix, be placed in 37 DEG C of water-bath 1h；
B. the sodium chloride solution of 200 μ l 5mol/L is subsequently adding, is centrifuged after mixing, centrifugal condition 13000rpm, 15min；
C. supernatant is taken, with phenol extraction 2 times, chloroform 1 time；
D. plus two volumes dehydrated alcohol, 1/10 volume, the potassium acetate of 3mol/L, pH8.0, -20 DEG C of preservation 1h；
E. above-mentioned solution is centrifuged, centrifugal condition 13000rpm, 15min abandon supernatant, precipitate washes 2 with 70% ethanol
It is secondary；
F. it is placed in after drying at room temperature, in being dissolved in 50 μ lTE solution (Shanghai Suo Laibao bio tech ltd), puts 4 DEG C of preservations standby
With.
9. according to claim 8, wherein described cracking formula of liquid is：40mmol/L Tris- acetic acid, 20mmol/L sodium acetates,
1mmol/L EDTA, 1% sodium lauryl sulphate (SDS), pH7.8.
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| CN201611043139.XA CN106636364B (en) | 2016-11-11 | 2016-11-11 | Multiplex PCR kit for rapidly identifying species of arowana and identification method thereof |
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| CN201611043139.XA CN106636364B (en) | 2016-11-11 | 2016-11-11 | Multiplex PCR kit for rapidly identifying species of arowana and identification method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113005203A (en) * | 2021-03-27 | 2021-06-22 | 中国水产科学研究院珠江水产研究所 | Microsatellite marking method for identifying paternity of scleropages formosus |
| CN114164282A (en) * | 2021-11-29 | 2022-03-11 | 中国水产科学研究院珠江水产研究所 | Primer for identifying genetic sex of double-wishbone and PCR (polymerase chain reaction) identification method |
Citations (1)
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| CN102286631A (en) * | 2011-09-14 | 2011-12-21 | 厦门出入境检验检疫局检验检疫技术中心 | Method for multiplex polymerase chain reaction (PCR) fast detection and five-Listeria-bacterium identification |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286631A (en) * | 2011-09-14 | 2011-12-21 | 厦门出入境检验检疫局检验检疫技术中心 | Method for multiplex polymerase chain reaction (PCR) fast detection and five-Listeria-bacterium identification |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113005203A (en) * | 2021-03-27 | 2021-06-22 | 中国水产科学研究院珠江水产研究所 | Microsatellite marking method for identifying paternity of scleropages formosus |
| CN114164282A (en) * | 2021-11-29 | 2022-03-11 | 中国水产科学研究院珠江水产研究所 | Primer for identifying genetic sex of double-wishbone and PCR (polymerase chain reaction) identification method |
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