CN114941034A - COI primer pair, kit and identification method for identifying cupfish and cupfish - Google Patents
COI primer pair, kit and identification method for identifying cupfish and cupfish Download PDFInfo
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Abstract
The invention provides a COI primer pair, a kit and an identification method for identifying a cupfish and a cupfish, belonging to the field of animal molecular genetics. The kit utilizes 1 pair of mitochondria COI sequence specific primers to carry out PCR amplification reaction on fish samples, after the reaction, the PCR amplification strips are found to have larger difference between the cudworted copper fishes and the copper fishes, and the cudworted copper fishes and the copper fishes can be directly identified through gel electrophoresis display. The invention designs 1 pair of primers in two fishes of cyprinid copper for PCR amplification and gel electrophoresis display to directly analyze and identify, and has the advantages of accurate experiment, reliable and rapid identification, simple and convenient operation and the like. The method can be used for rapidly identifying two species of the round-mouth copper fish and the copper fish, and provides technical support for germplasm identification of large-scale releasing of the round-mouth copper fish.
Description
Technical Field
The invention relates to a COI primer pair, a kit and an identification method for identifying a round-mouth copperfish and a copperfish, belonging to the field of animal molecular genetics.
Background
The mudskipper (Corei mu s guichenoti) belongs to Cypriniformes, gobianidae and coppers (Coreius), is mainly distributed in upstream water areas of Yangtze river, is a local important economic fish and a main fishing object, and is also an index species of a rare and specific fish protection area in the upstream of the Yangtze river. In recent years, with the influence of vigorous development and excessive fishing of hydropower stations at the upstream of the Yangtze river on ecological environment, an egg laying field of the round-mouth copper fishes is damaged, a migration channel is blocked, the population resource amount of the round-mouth copper fishes is rapidly reduced, species survival faces a huge threat, and the round-mouth copper fishes are listed in a Critical (CR) grade in 2016. Therefore, the work of enhancing the resource protection and recovery of the round-mouth coppers is urgent. At present, proliferation and releasing are considered to be one of the main ways for resource protection and ecological restoration of the round-mouth coppers.
In order to ensure the ecological safety of the release, the release type and source must be strictly controlled. In principle, the releasing seedlings are mainly local breeder seeds and first filial generation seedlings, and hybrid seeds, transgenic seeds, species with impure germplasm and the like which do not meet the ecological safety requirement cannot be put into the natural water area. In order to ensure the seed quality purity and the genetic quality of the released round-mouth copper fishes, the released offspring seeds need to be identified. The cyprinid family copperfish belongs to three fishes, namely round-mouth coppers (Coreus guichenoti), coppers (Coreus heterodon) and northern coppers (Coreus septiceriae), wherein the northern coppers are special fishes in the yellow river basin and are only distributed in the yellow river basin, so that the main interference species for releasing the round-mouth coppers in the Yangtze river basin are coppers, the round-mouth coppers and the coppers have similar morphological characteristics in the early development stage and are difficult to identify only from the morphology, and therefore a rapid identification method starting from the specificity of a genome is needed.
Cytochrome C oxidase subunit i (coi) is located on the mitochondrial genome, and is the gene with the largest molecular weight and the most conserved functional domains among 13 genes encoding proteins in the mitochondrial genome. Meanwhile, the COI gene can distinguish species with long differentiation time due to the slow evolution rate of the amino acid, and the COI gene sequence of the same species has enough variation and can distinguish the species with close relativity, so that the COI gene is widely used for molecular systematics research on different classification levels. Moreover, the COI gene is easy to amplify by a universal primer while ensuring enough variation, the 5' end sequence of most animals can be obtained, and the sequence has few insertions or deletions, so the COI gene is selected as a marker gene of DNA classification; meanwhile, the method has wide application prospect in species identification by utilizing the PCR technology. Therefore, the COI gene can be used for identifying the round-mouth coppers and the coppers.
Disclosure of Invention
In view of the above problems, the invention provides a COI primer pair, a kit and an identification method for identifying a round-mouth copper fish and a copper fish, the primer pair, the kit and the identification method can be used for rapidly identifying the round-mouth copper fish and the copper fish from a molecular level, and the method is simple and easy to operate and has high accuracy.
The technical scheme for solving the technical problems is as follows:
the invention provides a COI primer pair for identifying a cupfish and a cupfish, which consists of an upstream primer shown by SEQ ID NO. 1 and a downstream primer shown by SEQ ID NO. 2.
The invention also provides application of the COI primer pair in preparation of a kit for identifying the round-top copper fishes and the copper fishes.
The invention also provides a kit for identifying the round-mouth coppers and the coppers, which comprises the COI primer pair, Taq DNA polymerase, 10 XPCR Buffer, dNTP and MgCl 2 ，ddH 2 O。
The invention also provides a method for identifying the round-mouth copper fishes and the copper fishes, which comprises the following steps:
(1) extracting the genome DNA of a sample to be detected;
(2) amplifying the DNA template by using the genomic DNA extracted in the step (1) as a template and using the kit of claim 3.
(3) And (3) judging according to the amplification result in the step (2): if two bands are amplified, the DNA template comes from the round-mouth coppers; if a bright band is amplified, the DNA template is derived from the copper fish.
Preferably, the amplification system is as follows:
preferably, the concentration of the DNA template is 100-500 ng/. mu.L.
Preferably, the concentration of the pre-primer or the post-primer in the amplification system is 100-1000 pmol/. mu.L.
Preferably, the amplification procedure is: pre-denaturing at 93-95 deg.c for 3-5 min; denaturation at 93-95 deg.C for 25-35s, annealing at 55-60 deg.C for 30-50s, extension at 72 deg.C for 30-60s, and 30-40 cycles; extending for 5-10min at 72 ℃; storing at 4 deg.C.
Preferably, the amplification procedure is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
Advantageous effects
1. By using the mitochondrial COI gene as a template to design primers, the round-mouth coppers and the coppers with close genetic relationship can be identified, and the accuracy rate reaches 100%.
2. The kit can quickly and simply complete identification work, can simply identify the round-mouth coppers and the coppers without sequencing, and has the advantages of quick use, simple operation and the like; the method is suitable for germplasm identification such as releasing and the like and market supervision such as fishery enforcement, illegal trade and the like.
Drawings
FIG. 1 is a diagram showing the alignment of mitochondrial COI sequences of a Bractenopharyngodon idellus and a Bractenopharyngodon idellus.
FIG. 2 is a gel electrophoresis image of amplified fragments of 6 pairs of primers of mitochondrial COI in the DNA of Cultis rotundifolia and Cultis rotundifolia. Wherein M: DL1000 Marker; NC: and (5) negative control, wherein the serial numbers 1-6 are round-mouthed copper fish samples, and the serial numbers 7-12 are copper fish samples.
FIG. 3 is a gel electrophoresis of the amplified fragments of primers Cg1-F and Cg1-R in Cuprilus cupreum and Cuprilus cupreum. Wherein the numbers 1-10 are round-mouth copper fish samples, the numbers 11-20 are copper fish samples, and the rest are marked as the same as the figure 2.
FIG. 4 is a gel electrophoresis of the amplified fragments of primers Cg1-F and Cg1-R in round-nose copper fish and random mixed samples of copper fish. Wherein the numbers 1-16 are random round-mouth copper fish and copper fish samples, and the rest are marked as the same as the figure 2.
Detailed description of the preferred embodiments
The present invention will be described in detail with reference to the following drawings and examples, which are provided for illustration only and are not intended to be limiting.
Example 1 DNA sample extraction
The extraction of the genome DNA of the round-mouth copper fish and the copper fish adopts a DNA extraction kit (DP304) of Tiangen Biochemical technology (Beijing) Limited company, and the specific operation steps are as follows:
(1) respectively taking 0.5g of round-mouth copper fish and 0.5g of copper fish fin strip tissue, placing the round-mouth copper fish and the copper fish fin strip tissue in a grinding bowl, placing the round-mouth copper fish and the copper fish fin strip tissue in a 2mL sterile centrifuge tube, adding 1 × TE sterile buffer solution, and soaking for 12h at 4 ℃;
(2) taking out the fin-shaped tissue, putting the fin-shaped tissue into 352 mu L of sterile extraction buffer solution, gently mixing the fin-shaped tissue and the sterile extraction buffer solution, adding 40 mu L of 20% SDS and 10 mu L of 20mg/mL proteinase K (the final concentration is 400 mu g/mL), and mixing the mixture evenly;
(3) incubate at 56 ℃ for 4h, gently shake the tube every half hour to facilitate digestion. If the tissue is not easy to digest, the incubation time can be prolonged to 8h, or digestion is carried out overnight at 37 ℃;
(4) taking out the centrifuge tube after the tissue is completely digested, adding 300 mu L of 6M NaCI solution, and immediately and violently shaking for 30 s;
(5) centrifuging at 12000rpm for 10 min; discarding the precipitate, pouring the supernatant into a new centrifuge tube, centrifuging once under the same conditions, taking the supernatant, adding equal volume of pre-cooled isopropanol, and standing at 20 ℃ for more than 1 h;
(6) centrifuging at 12000rpm for 15min at 4 deg.C;
(7) discarding the supernatant, washing the precipitate with 70% ethanol solution once, and oven drying at 37 deg.C or naturally air drying; add 50. mu.L of sterilized ddH 2 O dissolving DNA, diluting to 100 ng/mu L after complete dissolution, and storing in a refrigerator at-20 ℃ for later use.
Example 2 screening of primer pairs
(1) Sequence analysis: the mitochondrial COI gene sequences of the cupfish and the cupfish are downloaded on an NCBI website, the COI sequence of the cupfish is SEQ ID NO:13, the COI sequence of the cupfish is SEQ ID NO:14, the sequences are submitted to MegAlign software for sequence comparison, sequence differences are analyzed, difference sites are found out, an analysis result (figure 1) shows that the similarity of the COI sequences of the cupfish and the cupfish is about 90%, base differences exist in a plurality of sites, and respective specific primers can be theoretically designed for distinguishing and identifying.
(2) Designing a primer: and (2) submitting the sequences to Primer6 software to design primers aiming at the differential sites analyzed in the step (1), wherein the Primer sequences are shown in a table 1. The primer sequences were then provided to Suzhou Jinzhi Biotechnology, Inc. for primer synthesis.
TABLE 1 specific primers based on mitochondrial COI gene sequences of Cultis rotundifolia and Cultis cupris
(3) And (3) PCR amplification: and (3) respectively carrying out PCR amplification on the DNA samples of the round-mouth copper fish and the copper fish obtained in the example 1 by using 6 pairs of primers in the step (2), wherein the enzyme used for amplification is Taq DNA polymerase.
The PCR amplification system and the amplification procedure are shown in Table 2 and Table 3, respectively.
TABLE 2 PCR amplification System
TABLE 3 PCR amplification procedure
(4) Gel electrophoresis: and (3) carrying out agarose gel electrophoresis on the PCR product obtained in the step (3) under the electrophoresis condition of 120V for 15min, wherein the result is shown in figure 2.
As shown in FIG. 2, the electrophoresis bands of the 6 pairs of primers are obviously different, wherein the first pair of primers Cg1-F and Cg1-R has two bands in the round-mouth copper fish and one bright band in the copper fish, and the band is about 700bp, which indicates that the pair of primers can identify the round-mouth copper fish and the copper fish; although the sizes of the round-mouth coppers and the coppers in the primer amplification of the 2 nd, 3 rd, 4 th, 5 th and 6 th pairs of primers are consistent with the designed sizes, the bands in the two fish samples have no obvious difference, which indicates that the round-mouth coppers and the coppers cannot be identified. Therefore, the Cg1-F and Cg1-R primer pairs can be used for amplifying the bands to distinguish the round-mouth copper fish from the copper fish.
Example 3 identification of the function of Cg1-F, Cg1-R
The Cg1-F and Cg1-R primer pairs selected in example 2 were further verified, and the sequences of the primers were SEQ ID NO:1, SEQ ID NO: 2.
10 round-mouth coppers and coppers genome DNAs are respectively and randomly extracted, and PCR amplification is carried out by using the primers, wherein the PCR reaction system and the PCR reaction program are shown in tables 2 and 3.
As shown in FIG. 3, the Cg1-F, Cg1-R primer pair amplified two bands on the Cultilus coitus DNA template and one bright band on the Cultilus coitus DNA template with a band size of about 700bp, which is consistent with the results of example 2, indicating that the Cg1-F, Cg1-R primer pair can be used to identify Cultix coitus and Cultix coitus.
Example 4 identification of fry
Randomly selecting 16 fries of the round-mouth coppers and the coppers, and performing amplification identification by using Cg1-F and Cg1-R primers.
Extracting genome DNA of 16 fish fries to be detected, wherein the method and the steps of DNA extraction are the same as those of the embodiment 1, then carrying out PCR amplification on Cg1-F and Cg1-R by using the primers determined in the embodiments 3 and 4, identifying the types of the fish fries, carrying out PCR amplification on the system and the program which are the same as those in the tables 2 and 3, respectively setting standard samples of the round-mouth copper fishes and the copper fishes as positive controls, and carrying out ddH (dichloro-diphenyl-trichloroethane) amplification on the standard samples of the round-mouth copper fishes and the copper fishes 2 O as a template as a negative control, the results are shown in FIG. 4.
As a result, as shown in FIG. 4, the electrophoretic bands of the random individuals of numbers 3, 4, 7, 9, 10, 12, 13, 15 were shown as two bands with darker brightness, which was consistent with the band of the round-necked cupfish standard, indicating that it was the round-necked cupfish. While the random individual samples numbered 1, 2, 5, 6, 8, 11, 14, 16 were bright single bands, consistent with the positive control for copper fish, indicating that they were copper fish.
Sequence listing
<110> Chinese sturgeon institute of Chinese changjiang three gorges group Limited
<120> COI primer pair, kit and identification method for identifying cupfish and cupfish
<130> 2022
<160> 14
<170> PatentIn version 3.5
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ctaagccagc ccggatcatt actaggtgac gatcaaatct ataatgttat cgtcactgcc 180
cacgccttcg taataatttt ctttatagta ataccaatcc ttattggtgg gtttggaaac 240
tgactcgtac cactaataat cggagcacct gatatagcat tcccacgaat aaataatata 300
agtttctgac ttctgccacc ctcattcctt cttctactag cttcttctgg agttgaagct 360
ggagccggaa caggatgaac agttatccac cacttgcagg taatcttgcc catgcaggag 420
catcagtaga cctaacaatt ttttcactac acctagcagg tgtctcatca atcttaggag 480
caattaactt cattaccaca accattaaca taaaacctcc agctatttct cagtaccaaa 540
cacccctttt gtatgagccg tacttgtaac agctgtactt ctccttctgt cactaccagt 600
tttagctgct ggaattacaa tgcttcttac agaccgtaat cttaatacta ctatcttcga 660
tccagcagga ggaggagacc caattctata tcaacattta ttctgattct ttggccaccc 720
agaagtctat attcttattt tacccggatt tggtattatt tcacatgttg tagcctacta 780
tgctggtaaa aaagaaccat ttggttatat aggaatagtt tgagctataa tagctattgg 840
cctcctagga tttattgttt gagcccatca tatgtttact gtaggaatag atgtagatac 900
ccgtgcctat tttacatccg caacaataat tattgctatc ccaacaggtg taaaagtatt 960
tagctgactt gctacgcttc atggcggctc aatcaaatga gaaacaccta tattatgagc 1020
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atcacttgac attgtcctcc atgacacata ttatgtagtt gcacacttcc actatgtatt 1140
atcaataggt gccgtatttg ctattatggc agctttcgtt cattgattcc cactattttc 1200
aggatatacc ctaaatgata cttgaacaaa aatccacttt ggggtaatat tcattggtgt 1260
aacctcacat ttttcccaca gcacttccta ggattagcag gaatgccacg acgatactct 1320
gactaccccg atgcctatgc cctatgaaat acagtatcat ctattggatc actaatctct 1380
ctagtggcag taatcatatt cctatttatt ctctgagagg ccttcgccgc taaacgggaa 1440
gtatcctcag tagaattaac tataacaaat gtagaatgac ttcacggctg tcctccacca 1500
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taagccagcc cggatcacta ctaggtgatg atcaaaattt ataatgttat cgttactgcc 180
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ctgactcgtg ccgctaataa ttgggagcac ccgatatggc attcccacga ataaataata 300
ataagtttct gacttttgcc accctcattc ctctattact agcctcttcc ggggttgaag 360
ctggggctgg gacaggatga acagttaccc accacttgca ggtaatcttg cccatgcagg 420
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ggcaattaac ttcatcacca caaccattaa tatgaaaccc ccagctattt cccaatacca 540
aacaccccct ctttgtgtgg gccgtacttg taacagctgt acttctcctt ctatcactac 600
cagctttagc tgccggaatt acaatgcttc ttacagatcg taatcttaat aaccacattc 660
tttgacccag cagggggagg agacccaatt ttgtatcaac acttattctg attcttcggc 720
catcccgaag tctatattct tattttaccc ggatttggca tcatttcaca cgttgtagcc 780
tactacgctg gtaaaaaaga gccatttggt tatataggaa tggtttgagc cataatagct 840
atcggccctc cttggattca ttgtctgagc ccatcatatg tttaccgtcg gaatagatgt 900
agacacccgt gcctatttta catccgcaac aataattatc gccatcccaa caggcgtaaa 960
agtatttagc tgactcgcca cgctccatgg gggctctatc aaatgagaaa cgcccatact 1020
atgagccctc ggattcattt tcctctttac ggtcggcggg ttgacaggga tcgttctagc 1080
taactcatca cttgacattg tcctccacga cacgtactat gtagttgcac acttccacta 1140
cgtactgtca ataggcgccg tatttgccat tatagcagct ttcgttcatt gattcccact 1200
attttcagga tataccctaa atgacacttg aacaaaaatc cactttggag taatatttat 1260
tggtgtaaac ctcacatttt tcccacaaca cttcctagga ttagcgggaa tgccacgacg 1320
atactctgat taccccgatg cctacgccct atgaaataca gtatcatcta ttgggatcac 1380
taatctccct ggtagcggta attatgttcc tatttattct ttgagaggcc ttcgccgcta 1440
aacgagaagt atcctcagta gaactaacta caacaaacgt agaatgactt cacggctgcc 1500
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Claims (9)
1. A COI primer pair for identifying a round-mouth coppers and a coppers is characterized by comprising the following components: the COI primer pair consists of an upstream primer shown by SEQ ID NO. 1 and a downstream primer shown by SEQ ID NO. 2.
2. The use of the COI primer pair of claim 1 in the preparation of a kit for identifying a cupfish and a cupfish.
3. The utility model provides an appraise round-mouth copper fish and kit of copper fish which characterized in that: comprises the COI primer pair of claim 1, Taq DNA polymerase, 10 XPCR Buffer, dNTP, MgCl2, ddH 2 O。
4. A method for identifying round-mouth copper fishes and copper fishes is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting genomic DNA of a sample;
(2) amplifying the DNA template by using the genomic DNA extracted in the step (1) as a template and adopting the kit of claim 3;
(3) and (3) judging according to the amplification result in the step (2): if two bands are amplified, the DNA template comes from the round-mouth coppers; if a bright band is amplified, the DNA template is derived from the copper fish.
5. The method for identifying cupfish and cupfish as claimed in claim 4, wherein: the amplification system is as follows:
10×PCR Buffer 2.5μL
dNTP（2.5mmol/L） 0.5 μL
MgCl2 1.5 μL
taq DNA polymerase 0.5. mu.L
0.1-1.0 mu L of pre-primer
Rear primer 0.1-1.0. mu.L
DNA template 0.5-2.0. mu.L
ddH 2 Make up to 20. mu.L of O.
6. The method for identifying Cuprilus amoena and Cuprilus amoena as claimed in any one of claims 4 and 5, wherein the concentration of the DNA template is 100ng/μ L and 500ng/μ L.
7. The method for identifying Cuprilus amoebous and Cuprilus amoebous as claimed in any one of claims 4 and 5, wherein the concentration of the pre-primer or the post-primer in the amplification system is 100-1000pmol/μ L.
8. The method for identifying the round-mouth coppers and the coppers as claimed in claim 4, wherein the amplification procedure is as follows: pre-denaturation at 93-95 deg.C for 3-5 min; denaturation at 93-95 deg.C for 25-35s, annealing at 55-60 deg.C for 30-50s, extension at 72 deg.C for 30-60s, and 30-40 cycles; extending for 5-10min at 72 ℃; storing at 4 ℃.
9. The method for identifying the round-mouth coppers and the coppers as claimed in claim 4 or 8, wherein the amplification procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
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| CN202210612127.3A CN114941034B (en) | 2022-05-27 | 2022-05-27 | COI primer pair, kit and identification method for identifying copper fish with round mouth |
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| CN114941034A true CN114941034A (en) | 2022-08-26 |
| CN114941034B CN114941034B (en) | 2023-10-13 |
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| CN202210612127.3A Active CN114941034B (en) | 2022-05-27 | 2022-05-27 | COI primer pair, kit and identification method for identifying copper fish with round mouth |
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