CN112646899A - Euphausia superba DNA bar code standard detection sequence, primer and application thereof - Google Patents
Euphausia superba DNA bar code standard detection sequence, primer and application thereof Download PDFInfo
- Publication number
- CN112646899A CN112646899A CN202110057521.0A CN202110057521A CN112646899A CN 112646899 A CN112646899 A CN 112646899A CN 202110057521 A CN202110057521 A CN 202110057521A CN 112646899 A CN112646899 A CN 112646899A
- Authority
- CN
- China
- Prior art keywords
- dna
- sequence
- euphausia superba
- primer
- detection sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a Euphausia superba DNA bar code standard detection sequence, a primer and application thereof, belonging to the field of molecular biology, wherein the sequence is SEQ ID NO: 1. the invention also provides a pair of primers for detecting the standard barcode detection sequence of the antarctic krill DNA in the claim 1, and application of the standard barcode detection sequence of the antarctic krill DNA in identification of the antarctic krill. And (3) carrying out nucleotide sequence determination on the PCR amplification product obtained by using the primer to the DNA of the euphausia superba, judging the species identification result according to the sequence comparison analysis result, and if the species identification result is identical to the species identification result shown as SEQ ID NO:1 is less than 0.02, and the sample to be detected can be judged to be the euphausia superba. The method can distinguish the antarctic krill from other krill belonging to the same genus.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a DNA barcode standard detection sequence and primer for euphausia superba and application thereof.
Background
Euphausia superba (Euphausia superba) is commonly known as Euphausia superba and belongs to the phylum Arthropoda, Crustacea, Malacostraca, Pandalales, Euphausiacea, Euphausiaceae, Euphausiacea, Euphausia, and Euphausia, which are crustaceans plankton living in the southern ocean. Antarctic krill is a large krill, and the adult can reach 42-65mm long, while the adult can reach 25-40 mm. The eyes are round, and the frontal angle is short and blunt, extending to the left and right of the middle part of the eyes. The skull and chest nail has an obvious neck pit. The dorsal concha has a pair of lateral teeth on its lower margin, but the lateral teeth are degenerated or absent in older males and some females. The abdominal joint has no back thorn. The male adaptor is elongated, curved and pointed. The curved base projection extends to the middle of the top projection, and the tail end of the curved base projection is in an irregular blade shape. The lateral protrusion is in a large hook shape. The aspect ratio of the greater whisker end segment is at least 7.
From an animal geography, antarctic krill is listed as a unique species of antarctic, and is distributed only around the antarctic continent. However, the density of distribution of the krill south Pole is not the same in the ocean areas surrounding the Pacific, Atlantic and Indian oceans of Antarctica, with the highest density being in the Atlantic ocean area and the Indian ocean being the second lowest density distribution of the Pacific ocean. And the krill species distributed in the Antarctic sea area are similar in morphology and difficult to distinguish by morphological characteristics. Currently, species identification of antarctic krill is limited to morphological identification, but there are few professionals who can identify the antarctic krill, so that the antarctic krill cannot be accurately identified in many regions. Therefore, it is important to develop an effective identification method for euphausia superba.
Disclosure of Invention
The invention aims to provide a DNA barcode standard detection sequence of euphausia superba and application thereof in species identification. The DNA barcode standard detection sequence of the euphausia superba provided by the invention is beneficial to realizing the molecular identification of the euphausia superba and can effectively shorten the identification time.
The invention is realized by the following technical scheme:
the antarctic krill DNA bar code standard detection sequence is shown as SEQ ID NO:1 is shown.
The invention also provides primers for detecting the standard detection sequence of the euphausia superba DNA barcode, which comprise a forward primer 5'-TTA TAC TTT ATT TTC GGT GCA TGA GC-3' and a reverse primer 5'-AAA TGT TGG TAG AGA ATA GGG TCA-3'.
The invention also provides application of the DNA barcode standard detection sequence of the euphausia superba in identification of the euphausia superba.
The specific identification method comprises the following steps:
1) separating and extracting DNA from a tissue sample to be detected;
2) performing PCR amplification by using the DNA obtained in the step 1) as a template and adopting the forward primer and the reverse primer;
3) and (3) carrying out nucleotide sequence determination on the PCR amplification product obtained in the step 2), judging a species identification result according to a sequence comparison analysis result, and if the species identification result is identical to the sequence shown in SEQ ID NO:1 is less than 0.02, and the sample to be detected can be judged to be the euphausia superba.
The forward primer in the step 2) is 5'-TTA TAC TTT ATT TTC GGT GCA TGA GC-3', and the reverse primer is 5'-AAA TGT TGG TAG AGA ATA GGG TCA-3'.
Further, the conditions of the PCR amplification reaction in step 2) are: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 41 deg.C for 2min, extension at 72 deg.C for 3min, performing 40 cycles, and final extension at 72 deg.C for 10 min.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a DNA barcode standard detection sequence for identifying euphausia superba, which realizes the rapid and accurate identification of euphausia superba and overcomes the following defects in the prior art: individuals with incomplete disability are basically not identified, species with similar morphology cannot be distinguished, and the identification of complete individuals requires professionals who are familiar with euphausia superba. Compared with the traditional morphological identification, the identification time is effectively shortened, and the identification method is accurate.
The method disclosed by the invention is accurate in detection, and can be used for distinguishing the Antarctic krill from other krills in the same genus.
In the identification method provided by the invention, the repeatability and the stability of a PCR reaction system and reaction conditions are high.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products in example 1.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
Example 1
The standard detection sequence of the euphausia superba DNA bar code of the embodiment is shown as SEQ ID NO:1 is shown.
Based on the sequences, primers for detecting the standard detection sequence of the euphausia superba DNA barcode are designed, and comprise a forward primer 5'-TTA TAC TTT ATT TTC GGT GCA TGA GC-3' and a reverse primer 5'-AAA TGT TGG TAG AGA ATA GGG TCA-3'.
The application of the DNA barcode standard detection sequence of the antarctic krill in the embodiment comprises the specific method of identifying the antarctic krill
1) Separating and extracting DNA from a tissue sample to be detected;
2) using the DNA obtained in the step 1) as a template, adopting a forward primer and a reverse primer to carry out PCR amplification,
3) and (3) carrying out nucleotide sequence determination on the PCR amplification product obtained in the step 2), judging a species identification result according to a sequence comparison analysis result, and if the species identification result is identical to the sequence shown in SEQ ID NO:1 is less than 0.02, and the sample to be detected can be judged to be the euphausia superba.
Example 2
This embodiment differs from example 1 in that: the PCR amplification reaction conditions in the step 2) are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 41 deg.C for 2min, extension at 72 deg.C for 3min, performing 40 cycles, and final extension at 72 deg.C for 10 min. The rest is the same as in example 1.
Example 3
1) Extracting DNA of a detection sample:
and selecting 10 detection samples, wherein S1-S6 are euphausia superba collected in the astronaut sea area, and S7-S10 are euphausia superba collected in the astronaut sea area. The long-armed euphausia superba and the antarctic euphausia superba are both of the krill genus species and are relatively close to each other.
About 30mg of muscle was taken from each sample to be tested, and DNA was extracted according to the instructions of the FastPuffpe DNA Isolation Kit genome extraction Kit (Biotech Co., Ltd., Nanjing Novevzan). Taking muscle tissue of a sample to be detected, putting the muscle tissue into a 1.5ml centrifuge tube, adding 600 mu l of Deparaffinization Solution, violently whirling for 5s, carrying out water bath at 56 ℃ for 6min, and violently whirling for 20 s; centrifuging at 14000 Xg for 2min, and discarding the supernatant; add 200. mu.l Buffer FTL and 20. mu.l Proteinase K working solution to the sample and vortex to mix well. Carrying out water bath at 56 ℃ for 1h until the sample is completely digested, and reversing and uniformly mixing for several times; water bath at 90 ℃ for 1 h; adding 200 mul of Buffer FL and 200 mul of absolute ethyl alcohol into the treatment solution, and uniformly mixing for 15s by vortex; placing the FFPE DNA adsorption column in a collecting tube, transferring the mixed solution to the adsorption column, and centrifuging at 10000 Xg for 60 s; discarding the filtrate, placing the adsorption column in a collecting tube, adding 500 μ l Buffer FW1 (added with ethanol) into the adsorption column, and centrifuging at 10000 × g for 60 s; discarding the filtrate, placing the adsorption column in a collecting tube, adding 650 μ l Buffer FW2 (added with ethanol) into the adsorption column, and centrifuging at 10000 × g for 60 s; discarding the filtrate, placing the adsorption column in a collecting tube, and centrifuging the empty column at 10000 Xg for 3 min; the column was transferred to a new 1.5ml centrifuge tube, uncapped for 3min (ethanol evaporated), and 15-50. mu.l of Elution Buffer was added to the center of the column. Standing at room temperature for 1min, and centrifuging at 10000 Xg for 1 min; the adsorption column was discarded and the DNA was stored at-20 ℃ and kept at-70 ℃ for a long period.
2) And (3) PCR amplification:
the PCR reaction system is 25 mul and consists of 1 mul of each upstream primer and downstream primer, 12.5 mul of 2 multiplied Taq Master Mix, 3 mul of template DNA and 7.5 mul of sterile water; the reaction procedure for PCR was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 41 deg.C for 2min, extension at 72 deg.C for 3min, performing 40 cycles, and final extension at 72 deg.C for 10 min.
3) Sequencing of test samples
The PCR product was a 650bp band (electrophoretogram shown in FIG. 1). The PCR products were sent to the bio-company for sequencing, resulting in 10 sequencing sample sequences S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10.
5) Alignment of Gene sequences
The genetic distance alignment of the Antarctic krill DNA barcode standard sequence (shown as SEQ ID NO:1 in the sequence table) and 10 sequencing sample sequences (S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10) by using the biological software MEGA X shows that the genetic distances of the Antarctic krill standard sequence after alignment with 6 Antarctic krill nucleotide sequences S1, S2, S3, S4, S5 and S6 from the ocean of astronauts are respectively 0.0022, 0.0133, 0.0066, 0.0044, 0.0066 and 0.0066 which are respectively less than 0.02, and the homology of the sequencing sequence S7, S8, S9, S10, S0.1617, S0.1729 and S8602 with 4 samples of Euphausia superba. All six samples of S1, S2, S3, S4, S5 and S6 were determined to be euphausia superba. The alignment results are shown in Table 1, ES is a standard sequence of DNA barcode of Euphausia superba (SEQ ID NO: 1); S1-S10 are the nucleotide sequences of 10 detection samples respectively.
TABLE 1
| ES | ||||||||||
| S1 | 0.0022 | |||||||||
| S2 | 0.0133 | 0.0111 | ||||||||
| S3 | 0.0066 | 0.0044 | 0.0156 | |||||||
| S4 | 0.0044 | 0.0022 | 0.0133 | 0.0066 | ||||||
| S5 | 0.0066 | 0.0044 | 0.0156 | 0.0089 | 0.0066 | |||||
| S6 | 0.0066 | 0.0044 | 0.0156 | 0.0089 | 0.0066 | 0.0000 | ||||
| S7 | 0.1701 | 0.1729 | 0.1675 | 0.1758 | 0.1758 | 0.1757 | 0.1757 | |||
| S8 | 0.1617 | 0.1646 | 0.1592 | 0.1674 | 0.1674 | 0.1673 | 0.1673 | 0.0340 | ||
| S9 | 0.1729 | 0.1758 | 0.1761 | 0.1787 | 0.1787 | 0.1786 | 0.1786 | 0.0066 | 0.0364 | |
| S10 | 0.1701 | 0.1729 | 0.1675 | 0.1758 | 0.1758 | 0.1757 | 0.1757 | 0.0202 | 0.0270 | 0.0225 |
Sequence listing
<110> research institute for aquatic products in yellow sea of China institute for aquatic science
<120> Euphausia superba DNA barcode standard detection sequence, primer and application thereof
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 650
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 1
ttatacttta ttttcggtgc atgagctggg atagtaggta cttcactaag attgattatt 60
cgagctgagt taggacaacc aggtagttta attggagatg accaaattta taatgttgta 120
gttacagcac atgcttttgt tataatcttc tttatggtaa taccaattat gattggtggc 180
tttggtaact ggcttgttcc actaatgcta ggagcccctg atatggcatt cccacgaata 240
aacaacataa gattttgatt gttaccccct tccttaactc tcttattagg aagaggttta 300
gtagaaagtg gggttggtac tgggtgaaca gtatacccac ctttatcagc aggaatcgct 360
catgctggag cctctgttga tataggaatc ttctcgcttc atattgccgg tgcttcttca 420
attttaggag ccgtaacttt tattacaact gtaattaata tacgatcagc aggtataact 480
atagaccgta ttccattatt tgtatggtca gtgtttatta cagctatcct acttctcctc 540
tctctcccgg ttctagctgg agcaattact atgcttctta cagatcgtaa tttaaatacc 600
tcattcttcg acccagccgg tggtggtgac cctattctct accaacattt 650
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttatacttta ttttcggtgc atgagc 26
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aaatgttggt agagaatagg gtca 24
<210> 4
<211> 629
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 4
attggtacat tatactttat tttcggtgca tgagctggga tagtaggtac ttcactaaga 60
ttgattattc gagctgagtt aggacaacca ggtagtttaa ttggagatga ccaaatttat 120
aatgttgtag ttacagcaca tgcttttgtt ataatcttct ttatggtaat accaattatg 180
attggtggct ttggtaactg gcttgttcca ctaatgctag gagcccctga tatggcattc 240
gcttgttcca ctaatgctag gagcccctga tatggcattc gcttgttcca ctaatgctag 300
gagcccctga tatggcattc ggttggtact gggtgaacag tatacccacc tttatcagca 360
ggaatcgctc atgctggagc ctctgttgat ataggaatct tctcgcttca tattgccggt 420
gcttcttcaa ttttaggagc cgtaaacttt attacaactg taattaatat acgatcagca 480
ggtataacta tagaccgtat tccattattt gtatgatcag tgtttattac agctatccta 540
cttctcctct ctctcccggt tctagctgga gcaattacta tgcttcttac agatcgtaat 600
ttaaatacct cattcttcga cccagccgg 629
<210> 5
<211> 590
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 5
cataaagata ttggtacatt atactttatt ttcggtgcat gagctggaat agtaggtact 60
tcactaagat tgattattcg agctgagtta ggacaaccag gtagtttaat tggagatgac 120
caaatttata atgttgtagt tacagcacat gcttttgtta taatcttctt tatggtaata 180
ccaattatga ttggtgggtt tggtaactgg cttgttccac taatgctagg agcccctgat 240
atggcattcc cacgaataaa caacataaga ttttgattgc tacccccttc cttaactctc 300
ttattaggaa gaggtttagt agaaagtggg gttggtactg gatgaacagt atacccacct 360
ttatcagcag gaattgctca tgctggagcc tctgttgata taggaatctt ctcgcttcat 420
attgccggtg cttcttcaat tttaggagcc gtaaacttta ttacaactgt aattaatata 480
cgatctgcag gtataactat agaccgtatt ccattatttg tatgatcagt gtttattaca 540
gctatcctac ttctcctctc tctcccggtt ctagctggag caattactat 590
<210> 6
<211> 608
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 6
attcgagctg agttaggaca accaggtagt ttaattggag atgaccaaat ttataatgtt 60
gtagttacag cacatgcttt tgttataatc ttctttatgg taataccaat tatgattggt 120
ggctttggta actggcttgt tccactaatg ctaggagccc ctgatatggc attcccacga 180
ataaacaaca taagattttg attgttaccc ccttccttaa ctctcttatt aggaagaggc 240
ttagtagaaa gtggggttgg tactgggtga acagtgtacc cacctttatc agcaggaatc 300
gctcatgctg gagcctctgt tgatatagga atcttctcgc ttcatattgc cggtgcttct 360
tcaattttag gagccgtaaa ctttattaca actgtaatta atatacgatc agcaggtata 420
actatagacc gtattccatt atttgtatga tcagtgttta ttacagctat cctacttctc 480
ctctctctcc cggttctagc tggagcaatt actatacttc ttacagatcg taatttaaat 540
acctcattct tcgacccagc cggtggtggt gaccctattc tctaccaaca tttattttga 600
ttttttgg 608
<210> 7
<211> 647
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 7
gcatgagctg ggatagtagg tacttcacta agattgatta ttcgagctga gttaggacaa 60
ccaggtagtt taattggaga tgaccaaatt tataatgttg tagttacagc acatgctttt 120
gttatgatct tctttatggt aataccaatt atgattggtg gctttggtaa ctggcttgtt 180
ccactaatgc taggagcccc tgatatggca ttcccacgaa taaacaacat aagattttga 240
ttgttacccc cttccttaac tctcttatta ggaagaggtt tagtagaaag tggggttggt 300
actgggtgaa cagtataccc acctttatca gcaggaatcg ctcatgctgg agcctctgtt 360
gatataggaa tcttctcgct tcatattgcc ggtgcttctt caattttagg agccgtaaac 420
tttattacaa ctgtaattaa tatacgatca gcaggtataa ctatagaccg tattccatta 480
tttgtatgat cagtgtttat tacagctatc ctacttctcc tctctctccc ggttctagct 540
ggagcaatta ctatgcttct tacagatcgt aatttaaata cctcattctt cgacccagcc 600
ggtggtggtg accctattct ctaccaacat ttattttgat tttttgg 647
<210> 8
<211> 635
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 8
aggtacttca ctaagattga ttattcgagc tgagttagga caaccaggta gtttaattgg 60
agatgaccaa atttataatg ttgtagttac agcacatgct tttgttataa tcttctttat 120
ggtaatacca attatgattg gtggctttgg taactggctt gttccactaa tgctaggagc 180
ccctgatatg gcattcccac gaataaacaa cataagattt tgattattac ccccttcctt 240
aactctctta ttaggaagag gtttagtaga aagtggggtt ggtactgggt gaacagtata 300
cccaccttta tcagcaggaa tcgctcatgc tggagcctct gttgatatag gaatcttctc 360
gcttcatatt gccggtgctt cttcaatttt aggagccgta aactttatta caactgtaat 420
taatatacga tcagcaggta taactataga ccgaattcca ttatttgtat gatcagtgtt 480
tattacagct atcctacttc tcctctctct cccggttcta gctggagcaa ttactatgct 540
tcttacagat cgtaatttaa atacctcatt cttcgaccca gccggtggtg gtgaccctat 600
tctctaccaa catttatttt gattttttgg tcacc 635
<210> 9
<211> 635
<212> DNA
<213> Euphausia superba (Eucheuma superba)
<400> 9
aggtacttca ctaagattga ttattcgagc tgagttagga caaccaggta gtttaattgg 60
agatgaccaa atttataatg ttgtagttac agcacatgct tttgttataa tcttctttat 120
ggtaatacca attatgattg gtggctttgg taactggctt gttccactaa tgctaggagc 180
ccctgatatg gcattcccac gaataaacaa cataagattt tgattattac ccccttcctt 240
aactctctta ttaggaagag gtttagtaga aagtggggtt ggtactgggt gaacagtata 300
cccaccttta tcagcaggaa tcgctcatgc tggagcctct gttgatatag gaatcttctc 360
gcttcatatt gccggtgctt cttcaatttt aggagccgta aactttatta caactgtaat 420
taatatacga tcagcaggta taactataga ccgaattcca ttatttgtat gatcagtgtt 480
tattacagct atcctacttc tcctctctct cccggttcta gctggagcaa ttactatgct 540
tcttacagat cgtaatttaa atacctcatt cttcgaccca gccggtggtg gtgaccctat 600
tctctaccaa catttatttt gattttttgg tcacc 635
<210> 10
<211> 513
<212> DNA
<213> Thysanoessa macrura
<400> 10
ttatatttca tttttggtgc atgagctggg atagtaggta cttcactaag attaattatt 60
cgagctgaat tgggacaacc aggtaggtta attggcgatg atcaaattta taatgttgta 120
gttacagcac atgcttttgt tataattttc tttatggtaa taccgattat aattggggga 180
tttggtaatt gacttgtacc tcttatatta ggagctccgg acatagcctt tccacgtata 240
aacaacataa ggttctggct tttacctcct tctttaacac tcttattagg tagtggtctt 300
gtagaaagag gtgtcggtac aggatgaaca gtttaccctc cattatcagc aggtattgca 360
catgctggtg cttcagttga tatgggtatc ttttctctcc atattgctgg tgcttcttct 420
attttgggag cagtaaattt cattaccact gttattaaca tgcgatctgc tggtataact 480
atagaccgta ttcctttatt tgtatggtct gtc 513
<210> 11
<211> 566
<212> DNA
<213> Thysanoessa macrura
<400> 11
cactttatat ttcatttttg gtgcatgagc tgggatagta ggtacttcac taagattaat 60
tattcgagct gaattgggac aaccaggtag gctaattggg gatgaccaaa tttacaatgt 120
tgtagttaca gcacatgctt ttgttataat tttctttatg gtaataccga ttataattgg 180
gggatttggt aattgacttg tacctcttat attaggagcc ccagacatag cctttccacg 240
tataaacaac ataagattct gacttttacc tccttcttta acactcttat tgggtagtgg 300
tcttgtagaa agaggtgtcg gtacaggatg aacagtttac cctccattat cagcaggtat 360
tgcacatgct ggtgcttcag tcgatatggg tatcttctct ctccatattg ctggtgcttc 420
ttctatttta ggagcagtaa attttattac cactgttatt aacatacgat ctgctggtat 480
aactatagac cgtattcctt tatttgtgtg gtctgtcttt attacagcta ttttactttt 540
attatcccta ccagttttag caggag 566
<210> 12
<211> 607
<212> DNA
<213> Thysanoessa macrura
<400> 12
gtacttcact aagattaatt attcgagctg aattgggaca accaggtagg ctaattggcg 60
atgatcaaat ttataatgtt gtagttacag cacatgcttt tgttataatt ttctttatgg 120
taataccgat tataattggg ggatttggta attgacttgt acctcttata ttaggagctc 180
cggacatagc ctttccacgt ataaacaaca taaggttctg gcttttacct ccttctttaa 240
cactcttatt aggtagtggt cttgtagaaa gaggtgtcgg cacagggtga acagtttacc 300
ctccattatc agcaggtatt gcacatgctg gtgcttcagt tgatatgggt atcttttctc 360
tccatattgc tggtgcttct tctattttgg gagcagtaaa tttcattacc actgttatta 420
acatgcgatc tgctggtata actatagacc gtattccttt atttgtatgg tctgtcttta 480
ttacagctat tttactttta ttatccctac cagttttagc aggagctatt actatattac 540
taacggaccg taatttgaat acttcttttt ttgatccagc aggaggggga gaccctattt 600
tatacca 607
<210> 13
<211> 607
<212> DNA
<213> Thysanoessa macrura
<400> 13
gtacttcact aagattaatt attcgagctg aattggggca accaggtagg ctaattggcg 60
atgatcaaat ttataatgtt gtagttacag cacatgcttt tgttataatt ttctttatgg 120
taataccgat tataattggg ggatttggta attgacttgt acctcttata ttaggagctc 180
cagacatagc ctttccacgt ataaacaaca taagattctg acttttacct ccttctttaa 240
cactcttatt aggtagtggt cttgtagaaa gaggtgtcgg tacaggatga acagtttacc 300
ctccattatc agcaggtatt gcacatgctg gtgcttcagt tgatatgggt atcttttctc 360
tccatattgc tggtgcttct tctattttgg gagcagtaaa tttcattacc actgttatta 420
atatacgatc tgctggcata actatagacc gtattccttt atttgtgtgg tctgtcttta 480
ttacagctat tttactttta ttatccctac cagttttagc aggagctatt actatactac 540
taacggaccg taatttgaat acttcttttt ttgatccagc aggaggggga gaccctattt 600
tatacca 607
Claims (4)
1. A Euphausia superba DNA barcode standard detection sequence is characterized in that the sequence is SEQ ID NO: 1.
2. a pair of primers for detecting the Euphausia superba DNA barcode standard detection sequence of claim 1, wherein the primers comprise a forward primer 5'-TTA TAC TTT ATT TTC GGT GCA TGA GC-3' and a reverse primer 5'-AAA TGT TGG TAG AGA ATA GGG TCA-3'.
3. The application of the antarctic krill DNA barcode standard detection sequence in the antarctic krill identification according to claim 1, characterized in that the application method is
1) Separating and extracting DNA from a tissue sample to be detected;
2) performing PCR amplification by using the DNA obtained in the step 1) as a template and adopting a forward primer and a reverse primer; the forward primer is 5'-TTA TAC TTT ATT TTC GGT GCA TGA GC-3', and the reverse primer is 5'-AAA TGT TGG TAG AGA ATA GGG TCA-3';
3) and (3) carrying out nucleotide sequence determination on the PCR amplification product obtained in the step 2), judging a species identification result according to a sequence comparison analysis result, and if the species identification result is identical to the sequence shown in SEQ ID NO:1 is less than 0.02, and the sample to be detected can be judged to be the euphausia superba.
4. The use according to claim 3, wherein the PCR amplification reaction of step 2) is performed under the following conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 41 deg.C for 2min, extension at 72 deg.C for 3min, performing 40 cycles, and final extension at 72 deg.C for 10 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110057521.0A CN112646899A (en) | 2021-01-15 | 2021-01-15 | Euphausia superba DNA bar code standard detection sequence, primer and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110057521.0A CN112646899A (en) | 2021-01-15 | 2021-01-15 | Euphausia superba DNA bar code standard detection sequence, primer and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112646899A true CN112646899A (en) | 2021-04-13 |
Family
ID=75368382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110057521.0A Pending CN112646899A (en) | 2021-01-15 | 2021-01-15 | Euphausia superba DNA bar code standard detection sequence, primer and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112646899A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130260380A1 (en) * | 2010-11-05 | 2013-10-03 | Diana Hall | Method for detecting the presence of a dna minor contributor in a dna mixture |
| CN109055404A (en) * | 2018-07-25 | 2018-12-21 | 江汉大学 | A kind of band south loach DNA bar code sequence and its application |
-
2021
- 2021-01-15 CN CN202110057521.0A patent/CN112646899A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130260380A1 (en) * | 2010-11-05 | 2013-10-03 | Diana Hall | Method for detecting the presence of a dna minor contributor in a dna mixture |
| CN109055404A (en) * | 2018-07-25 | 2018-12-21 | 江汉大学 | A kind of band south loach DNA bar code sequence and its application |
Non-Patent Citations (2)
| Title |
|---|
| W P GOODALL-COPESTAKE等: "Swarms of diversity at the gene cox1 in Antarctic krill", 《HEREDITY》 * |
| 谌微等: "基于线粒体COI基因序列的磷虾目分子系统进化", 《中国水产科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Walker et al. | Comparison of the 5.8 S rRNA gene and internal transcribed spacer regions of trichomonadid protozoa recovered from the bovine preputial cavity | |
| Guo et al. | Genetic characterization of Toxoplasma gondii strains by random amplified polymorphic DNA polymerase chain reaction | |
| Eszterbauer et al. | Molecular characterization of Sphaerospora molnari (Myxozoa), the agent of gill sphaerosporosis in common carp Cyprinus carpio carpio | |
| Sun et al. | Detection of genetic relationships among four Artemia species using randomly amplified polymorphic DNA (RAPD) | |
| CN110607359A (en) | Patinopecten yessoensis female specific marker combination and application | |
| CN113462787B (en) | Male molecular marker of optical barb cheilus and application thereof | |
| Horbańczuk et al. | A search for sequence similarity between chicken (Gallus domesticus) and ostrich (Struthio camelus) microsatellite markers | |
| CN100415884C (en) | DNA molecular marking method for researching fish genetic relation | |
| CN112210619B (en) | Primer pair for detecting bicuspid plum blossom yeast and application thereof | |
| CN106636319B (en) | Molecular biological method for rapidly identifying gibbon and northern Baijiagibbon | |
| Tan et al. | Sequence variation in the cytochrome oxidase subunit I and II genes of two commonly found blow fly species, Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart)(Diptera: Calliphoridae) in Malaysia | |
| CN112646899A (en) | Euphausia superba DNA bar code standard detection sequence, primer and application thereof | |
| Watthanakaiwan et al. | Molecular characterization of the ribosomal DNA unit of Sarcocystis singaporensis, Sarcocystis zamani and Sarcocystis zuoi from rodents in Thailand | |
| Hänni et al. | Molecular typing of Neolithic human bones | |
| CN110607376A (en) | Patinopecten yessoensis living body sex identification method based on DNA molecular marker | |
| CN113881786B (en) | Primer for identifying pseudobagrus ussuriensis, pseudobagrus ussuriensis and individuals with forward and reverse hybridization of pseudobagrus ussuriensis and pseudobagrus ussuriensis | |
| CN111500774B (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| CN106591426B (en) | COI gene standard complete sequence and molecular identification method of great brow apes | |
| KR100843575B1 (en) | Gene of a parasite causing death of Halocynthia roretzi and method for diagnosing the parasite | |
| Kim et al. | Validation of a novel one-step reverse transcription polymerase chain reaction method for detecting viral haemorrhagic septicaemia virus | |
| CN114480601A (en) | Molecular marker, primer, method and application for identifying genetic sex of hippocampus kelloggi | |
| Park et al. | Mitochondrial cytochrome b sequence variation in Korean salmonids | |
| CN110616270A (en) | COI gene sequence-based molecular identification method of beta and beta | |
| Khaier et al. | Molecular characterization of Eimeria acervulina in broiler chickens | |
| CN111315764A (en) | Petal purpurin and coding gene thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210413 |