CN108642185A - A kind of molecular labeling and application for tiger spot cuttlefish with quasi- mesh cuttlefish species identification - Google Patents
A kind of molecular labeling and application for tiger spot cuttlefish with quasi- mesh cuttlefish species identification Download PDFInfo
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- CN108642185A CN108642185A CN201810270759.XA CN201810270759A CN108642185A CN 108642185 A CN108642185 A CN 108642185A CN 201810270759 A CN201810270759 A CN 201810270759A CN 108642185 A CN108642185 A CN 108642185A
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Abstract
The present invention relates to a kind of molecular labeling can be used for tiger spot cuttlefish and quasi- mesh cuttlefish species identification and application, belong to sibling species identification technology, the method for the nearly edge species identification of specifically a kind of cuttlefish.Specially using tiger spot cuttlefish and quasi- mesh cuttlefish genomic DNA as template, PCR amplification, and then differentiation cuttlefish species different using Tm value difference of the amplified production in high-resolution melting curve (HRM) are carried out according to the sequence label design primer of tiger spot cuttlefish and quasi- mesh cuttlefish species gene group；It is capable of the identification of quick, economy, simplicity carried out between species using the present invention, accuracy rate is 100% compared with sequence label sequencing result.
Description
Technical field
The invention belongs to cuttlefish species identification technical fields more particularly to one kind can be used for tiger spot cuttlefish and quasi- mesh cuttlefish kind
Between the molecular labeling identified and application.
Background technology
Tiger spot cuttlefish (Sepia pharaonis) be subordinate to Mollusca (Mollusca), Sepiida (Sepioidea),
Sepiidae (Sepiidae), Sepia (Sepia).It is distributed in the Pacific Tropical Ocean Area of India, from offshore to depth of water 100m's
It is found in waters.Quasi- mesh cuttlefish (Sepia lycidas) is subordinate to Cephalopoda (Cephalopoda), Sepiida
(Sepioidea), Sepiidae (Sepiidae), Sepia (Sepia) are that water warm stronger bottom in shallow sea is dwelt kind, inhabit big
The offshore waters of shelf area 15~100m depth of waters, are distributed in the Indian Ocean-Western Pacific.Both nutritive value it is good and
With certain medical value；Tiger spot cuttlefish and quasi- mesh cuttlefish belong to Sepiidae Sepia, have point in coastal area of southeastern China
Cloth, Phylogenetic Analysis is research shows that tiger spot cuttlefish is closer with quasi- mesh cuttlefish affiliation, and two kinds of cuttlefishes are in growth traits
Have many similar, depend merely on morphology and be not easily distinguishable, especially in the young period of breeding production, it more difficult to distinguish.Therefore, selection has
The method of effect is then of great significance to distinguish tiger spot cuttlefish and quasi- mesh cuttlefish；In recent years, with the development of molecular biology,
It identifies that species have been increasingly becoming a kind of trend using molecular biology method, and is received by most of biologist.
From the third generation New molecular marker technology that American scholar Lander was proposed in 1996 after RFLP, SSR --- since SNP,
The detection of SNP and parting have become the key technology of genetics research, and SNP is referred in certain biological Different Individual DNA sequence dna
Polymorphism caused by the polymorphism that single nucleotide acid point mutation generates, including displacement, transversion, insertion or missing.High-resolution
Melting curve method is the difference based on different nucleic acid physical properties, and PCR reaction systems are added in fluorescent dye, are contaminated when containing fluorescence
The DNA of material solves supination, can collect fluorescence information and form melting curve.When some base changes in DNA sequence dna, melt
Curve will occur to change accordingly, can be analyzed hereditary information by software, have efficient, accurate, high pass
The advantages that amount, low cost.Currently, HRM technologies have been applied to the identification of known type, Mutation Screening, DNA methylation point
Analysis, the fields such as microbial molecular parting and identification and rna editing are the hot technologies in molecule diagnosis.
Invention content
A technical problem to be solved by this invention is present situation offer one kind for the prior art for tiger spot cuttlefish
With the molecular labeling of quasi- mesh cuttlefish species identification.
Another technical problem to be solved by this invention is a kind of above-mentioned point of utilization of present situation offer for the prior art
Son marks to distinguish the application of tiger spot cuttlefish and quasi- mesh cuttlefish species identification.
Another technical problem to be solved by this invention is a kind of above-mentioned point of utilization of present situation offer for the prior art
The method that son marks to distinguish tiger spot cuttlefish and quasi- mesh cuttlefish species identification.
Technical solution is used by the present invention solves above-mentioned technical problem：This can be used for tiger spot cuttlefish and quasi- mesh cuttlefish kind
Between the SNP marker identified, it is characterised in that：The SNP marker
Forward primer F1 is 5'ACGGGCGATATGTACGCG 3'；
Reverse primer F2 is 5'CTAATATGTCAAGTCAAAATGCAGC 3'.
The present invention provides a kind of above-mentioned SNP marker of utilization molten based on high resolution to solve second technical problem
Solution curve identifies tiger spot cuttlefish and the application in quasi- mesh cuttlefish.
Further, the SNP in the tiger spot cuttlefish target amplified fragments is：
ACGGGCGATATGTACGCGCTATAAAACCTGATTCATATAAACATATTAATTATTTACATT
ACTATTAAGTCCAATTTCAAAAATACATTTACATTATTTAATCCAATTTAAAATTATAAA
TCGTAGCTCATTCTATTTTCACCTTTCCTATTAGCTGCATTTTGACTTGACATATTAG。
Further, the SNP in the quasi- mesh cuttlefish target amplified fragments is：
ACGGGCGATATGTACGCGCTATAAAACCTGATTCATATAAACATATTAATTATTTACATT
ACTATTAAGTCCAATTTCAAAAATAC-TTTACATTACTTAATCCATTTAAAAATTATAAAT AGTAGCTCATT-
TATAATAACCTTTTTTATTAGCTGCATTTTGACTTGACATATTAG。
The present invention for solve third technical problem provide it is a kind of based on high-resolution solubility curve identify tiger spot cuttlefish and
The method of quasi- mesh cuttlefish, it is characterised in that：
Using tiger spot cuttlefish and quasi- mesh cuttlefish genomic DNA as template, according to the genome of tiger spot cuttlefish and quasi- mesh cuttlefish
Sequence label design primer carries out PCR amplification, different in turn using Tm value difference of the amplified production in high-resolution melting curve
It distinguishes；Using cuttlefish genomic DNA to be identified as template, PCR amplification is carried out according to the gene order design primer of cuttlefish, is utilized
Tm value difference of the amplified production in high-resolution melting curve is different and then distinguishes the close species of cuttlefish.
Further, the PCR product obtained by above-mentioned amplification is added denaturation treatment after nucleic acid saturable dye, is cooled to
Room temperature carries out HRM measurement.
Compared with prior art, the invention has the advantages that：
1, method is simple and practicable；It need not be sequenced, directly by the Tm values of amplified fragments come the area in reaction dna level
Different plant species are not distinguished；
2, result is clearly intuitive；Without progress sequence alignment, the difference of DNA level is with straight between different plant species
The peak figure form of sight shows, and easily distinguishes and distinguishes；
3, accurate and effective；Identification method of the present invention is had using high-resolution melting curve (HRM) differentiates single nucleotide acid
The ability of difference can accurately reflect the difference on DNA level so that qualification result is accurate and reliable, compared with sequencing result
Accuracy rate is 100%；
4, application is extensive；It is different from sequencing, it need not be by all individual sequencing analysis one by one to be identified, in of the invention
One group of primer can carry out species identification without redesigning primer on infinitely how similar individual；The present invention passes through offer
Differentiate tiger spot cuttlefish and quasi- mesh cuttlefish SNP primers, Genotyping has successfully been carried out to two kinds of cuttlefishes by HRM technologies, from divide
Sub- level explores the gene fragment order difference of tiger spot cuttlefish and quasi- mesh cuttlefish, establishes tiger spot cuttlefish and quasi- mesh cuttlefish
Molecular identification method, secondly, the present invention are an open system, are had wide range of applications；It is close to cannot be only used for specific several cuttlefishes
The identification of edge species, as long as can be reflected using this method based on the cuttlefish species that the difference on DNA level can be distinguished
It is fixed.
Description of the drawings
Fig. 1 be the nearly edge species identification high-resolution melting curve figure of Sepia cuttlefish provided in an embodiment of the present invention (wherein
Abscissa is temperature DEG C), ordinate is the logarithm of fluorescence intensity；Abscissa corresponding to the peak of every curve is
The Tm values of the corresponding PCR product of the curve)；
Fig. 2 is that the standardization that the nearly edge species identification high-resolution of Sepia cuttlefish provided in an embodiment of the present invention melts is bent
Line；
Fig. 3 is the nearly edge species of Sepia cuttlefish provided in an embodiment of the present invention in the molten of standardization and different temperatures
Solution curve peak.
Specific implementation mode
Below by way of accompanying drawings and embodiments are combined, the invention will be further described.
The present invention introduces high-resolution melting curve side during being distinguished by nucleic acid sequence and carry out species identification
Difference of the different plant species on DNA level is only converted to more intuitive melting curve by method with one couple of PCR primers.
Its step is：
A, DNA is extracted
Genomic DNA is extracted using traditional phenol chloroform-isoamyl alcohol method, agarose gel electrophoresis detects DNA sample
Quality, NANODROP 2000 (Thermo Scientific, USA) measures DNA stock solution qualities and concentration, and is diluted to 50ng/
The concentration of ul is for use.
B, acquisition and sequence alignment analysis of the species identification according to sequence
It is soft using AlignX by obtaining the mtDNA sequence overall length of tiger spot cuttlefish and quasi- mesh cuttlefish from ncbi database
Part carries out sequence alignment, finds base difference present in two kinds of cuttlefishes；From containing SNP site sequence, selection meets primer
The sequence of design carries out SNP primer design using Premier5.0；
C, design of primers and screening
Design of primers should meet the following conditions：Target amplification sequence is between 50bp-150bp, to keep melting temperature different,
There are the differences of GC base contents between sequence；Annealing temperature (Tm) should be between 50 DEG C -60 DEG C；It should be use up between positive anti-primer
Amount avoids mispairing, hairpin structure and primer dimer；Primer is synthesized by Shanghai Sheng Gong engineering finites responsible company.Random selection 5
A corresponding cuttlefish DNA sample is template, and preliminary screening, choosing are carried out to primer using agarose gel electrophoresis technology is used
Bright, the primer progress HRM analyses of single band can be amplified by selecting
D, PCR amplification and high-resolution melting curve (HRM) analysis
Using the genomic DNA of target cuttlefishes to be identified as template, PCR amplification is carried out using above-mentioned primer；PCR system
Using cuttlefish genomic DNA as template, to adjust DNA concentration to 50ng/ μ l, the primer after screening is used for the HRM of target fragment
Analysis.In LightPCR amplification and product are carried out on real-time quantitative analysis instrument (Roche Diagnostics)
Melting curve analysis.Reaction uses LightHRM kits.
PCR system：20μL1×Light480HRM Master Mix with Resodye(Roche
Diagnostics), each 1 μ L of positive anti-primer, the DNA profiling of 1 μ L 100ng/ μ L, 2.8 μ L MgCl2, moisturizing to 20 μ L.
PCR amplification program is：95 DEG C (pre-degeneration 10min), 95 DEG C (denaturation 30sec), Tm (renaturation 30sec), 72 DEG C (are prolonged
Stretch 30sec) denaturation, renaturation, extend after three steps recycle 45 times altogether, solubility curve fluorescent collecting：95 DEG C denaturation 30seconds, 65
DEG C annealing 30seconds, 95 DEG C heating 30seconds.
After PCR amplification, product directly existsReal-time quantitative analysis instrument (Roche
Diagnostics the operation of HRM is carried out on) automatically, 1 DEG C is often increased and acquires fluorescence 25 times.It usesAbove certainly
1.5 softwares of Tm Calling and Gene Scanning Software of band carry out the analysis of Tm values and Genotyping.In addition, with
It can be directed to the optimal canonical sequence of different plant species by unrestricted choice as needed in the foundation sequence of species identification, can be COI
Gene order or other meet the sequence of biomarker feature.
E, sequence verification
For the SNP site of the apparent parting of energy, randomly chooses 5 corresponding PCR products of each genotype and is sequenced,
Sequencing result is then subjected to sequence alignment, the base composition difference between observing.
Embodiment 1
The identification of tiger spot cuttlefish and quasi- mesh cuttlefish in Sepia：
A, acquisition of the species identification according to sequence：It is complete by obtaining above-mentioned Sepia mtDNA sequence from ncbi database
It is long, sequence alignment is carried out using AlignX softwares, base difference segment present in two kinds of cuttlefishes is found, as species identification
According to sequence；
B, design of primers：Selection can obviously distinguish the sequence of target species in above-mentioned Sepia mitochondria full length sequence
PCR amplification primer is designed in region in the area using Primer Premier5 softwares, and primer extension product is in different plant species
Between should have apparent Tm value differences different, be mainly reflected in that C, T content is different.
Forward primer F1 is：5'ACGGGCGATATGTACGCG 3'；
Reverse primer F2 is：5'CTAATATGTCAAGTCAAAATGCAGC 3'.
C, PCR amplification：Using the genomic DNA of cuttlefish sample to be identified as template, PCR amplification is carried out using above-mentioned primer.
PCR amplification program according to：94 DEG C (pre-degeneration 10min), 94 DEG C (denaturation 15sec), 53 DEG C of Tm (renaturation 15sec), 72 DEG C (are prolonged
Stretch 30sec) denaturation, renaturation, extends and extend 5min after three steps recycle 35 times altogether after 72 DEG C, 4 DEG C (cooling) end.
D, high-resolution melting curve (HRM) is analyzed：Above-mentioned product is directly run on Lightscanner96 platforms
HRM collects the fluorescent signal data between 55 DEG C -95 DEG C.After using the analysis software carried on platform by fluorescence signal
It is converted to melting curve peak figure；The nearly edge of different Sepias is distinguished according to the difference of the corresponding Tm values of the peak value of different curves
Species (referring to Fig. 1).
Fig. 2 is through LightUpper Gene Scanning resume modules analysis, obtains standardization melting curve
View can preferably reflect that tiger spot cuttlefish changes with the different caused peak figure of quasi- mesh cuttlefish Tm value differences；
Fig. 3 is melting curve peak figure in the case of standardization and different temperatures, and Fig. 2 Fig. 3 is for preferably reflecting tiger spot
Peak figure variation caused by cuttlefish and quasi- mesh cuttlefish Tm value differences are different.
F, it respectively takes 30 wild quasi- mesh cuttlefishes and tiger spot cuttlefish as sample respectively, is detected after extracting DNA, tested
As a result as follows：
As seen from Figure 1：Melting curve is polymerized to 2 beams respectively, and 2 kinds of different Tm values are presented respectively, wild in counter sample
Quasi- mesh cuttlefish and tiger spot cuttlefish；Type A is that quasi- its TM value of mesh cuttlefish is 74.93 DEG C, and type B is that tiger spot cuttlefish its TM value is
76.12 DEG C, two groups of samples are significantly distinguished, it was demonstrated that the site is for effectively identifying both species.
E, sequence verification
For the SNP site of the apparent parting of energy, randomly chooses each corresponding PCR product of genotype curve and is sequenced,
The sequencing result of various genotype is compared, the base composition difference between observing, such as the following table 1.
1 sequence of table and comparison result：It shows and is present in tiger spot cuttlefish and quasi- mesh cuttlefish target in two kinds of Sepias
SNPs in amplified fragments, respectively SEQ1 and SEQ2, such as following table.
Meanwhile the base composition difference between observing, such as the following table 2, it shows and is present in the expansion of both cuttlefish targets
The SNPs increased in segment detects 11 SNP sites altogether in the differential fragment of this 178bp, wherein there is 3 base transitions
Site, 6 transversion sites, 2 base deletion sites, sequencing sequence comparison result specify the sequence difference between species.
Table 2：
Sequence table
<110>University Of Ningbo
<120>A kind of molecular labeling and application for tiger spot cuttlefish with quasi- mesh cuttlefish species identification
<130> Not published yet
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 178
<212> DNA
<213> Sepia pharaonis
<400> 1
acgggcgata tgtacgcgct ataaaacctg attcatataa acatattaat tatttacatt 60
actattaagt ccaatttcaa aaatacattt acattattta atccaattta aaattataaa 120
tcgtagctca ttctattttc acctttccta ttagctgcat tttgacttga catattag 178
<210> 2
<211> 176
<212> DNA
<213> Sepia lycidas
<400> 2
acgggcgata tgtacgcgct ataaaacctg attcatataa acatattaat tatttacatt 60
actattaagt ccaatttcaa aaatacttta cattacttaa tccatttaaa aattataaat 120
agtagctcat ttataataac cttttttatt agctgcattt tgacttgaca tattag 176
Claims (5)
1. a kind of SNP marker for tiger spot cuttlefish and quasi- mesh cuttlefish species identification, it is characterised in that：The SNP molecules
The forward primer F1 of label is 5'ACGGGCGATATGTACGCG3'；Reverse primer F2 is 5'
CTAATATGTCAAGTCAAAATGCAGC3'。
2. a kind of SNP marker according to claim 1 based on high resolution solubility curve identification tiger spot cuttlefish with
Application in quasi- mesh cuttlefish.
3. SNP marker according to claim 2 is based on high resolution solubility curve identification tiger spot cuttlefish and quasi- mesh
Application in cuttlefish, it is characterised in that：SNP in the tiger spot cuttlefish target amplified fragments is：
ACGGGCGATATGTACGCGCTATAAAACCTGATTCATATAAACATATTAATTATTTACATTACTATTAAGTCCAATTT
CAAAAATACATTTACATTATTTAATCCAATTTAAAATTATAAATCGTAGCTCATTCTATTTTCACCTTTCCTATTAG
CTGCATTTTGACTTGACATATTAG。
4. SNP marker according to claim 2 is based on high resolution solubility curve identification tiger spot cuttlefish and quasi- mesh
Application in cuttlefish, it is characterised in that：SNP in the quasi- mesh cuttlefish target amplified fragments is：
ACGGGCGATATGTACGCGCTATAAAACCTGATTCATATAAACATATTAATTATTTACATTACTATTAAGTCCA
ATTTCAAAAATACTTTACATTACTTAATCCATTTAAAAATTATAAATAGTAGCTCATT-
TATAATAACCTTTTTTATTAGCTGCATTTTGACTTGACATATTAG。
5. a kind of method for identifying tiger spot cuttlefish and quasi- mesh cuttlefish based on high-resolution solubility curve, it is characterised in that：With tiger spot
Cuttlefish is template with quasi- mesh cuttlefish genomic DNA, is drawn according to tiger spot cuttlefish and the sequence label design of the genome of quasi- mesh cuttlefish
Object carries out PCR amplification, and then differentiation different using Tm value difference of the amplified production in high-resolution melting curve；With cuttlefish to be identified
Genomic DNA is template, PCR amplification is carried out according to the gene order design primer of cuttlefish, using amplified production in high-resolution
Tm value differences in melting curve are different and then distinguish the close species of cuttlefish.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102758009A (en) * | 2012-06-08 | 2012-10-31 | 中国科学院海洋研究所 | Method for identifying related species of oyster |
| CN104046683B (en) * | 2013-03-13 | 2015-06-10 | 中国科学院海洋研究所 | Method for discriminating two closely-related species of shellfish or identifying their hybrid generation |
-
2018
- 2018-03-29 CN CN201810270759.XA patent/CN108642185A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102758009A (en) * | 2012-06-08 | 2012-10-31 | 中国科学院海洋研究所 | Method for identifying related species of oyster |
| CN104046683B (en) * | 2013-03-13 | 2015-06-10 | 中国科学院海洋研究所 | Method for discriminating two closely-related species of shellfish or identifying their hybrid generation |
Non-Patent Citations (2)
| Title |
|---|
| LAURE BONNAUD等: ""Morphological character evolution and molecular trees in sepiids (Mollusca: Cephalopoda): is the cuttlebone a robust phylogenetic marker?"", 《BIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY》 * |
| 温旺荣: "《临床分子诊断学》", 31 May 2015 * |
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Application publication date: 20181012 |