CN108315434A - A kind of red ear tortoise specific primer and its application - Google Patents
A kind of red ear tortoise specific primer and its application Download PDFInfo
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- CN108315434A CN108315434A CN201810190950.3A CN201810190950A CN108315434A CN 108315434 A CN108315434 A CN 108315434A CN 201810190950 A CN201810190950 A CN 201810190950A CN 108315434 A CN108315434 A CN 108315434A
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- 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
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Abstract
The invention discloses a kind of red ear tortoise specific primer and its applications.The nucleotide sequence such as SEQ ID NO of the red ear tortoise specific primer of the present invention:Shown in 1~2.The red ear tortoise specific primer of the present invention can be used for PCR and react and carry out electrophoresis detection, by the presence or absence of gained purpose band, quickly distinguish red ear tortoise and other 11 kinds of Chelonians being found in Chinese field.The present invention is by providing red ear tortoise specific primer, and establish the effective molecular detecting method for distinguishing red ear tortoise and other 11 kinds of Chelonians, effective means is provided for the quick identification of the red ear tortoise of Invasive Alien Species, this method is of less demanding to laboratory apparatus and consumptive material, and good technical support is provided to investigating the distribution of red ear tortoise field using environment DNA.
Description
Technical field
The invention belongs to species identification technical fields, it is more particularly related to a kind of for quickly distinguishing red ear
The red ear tortoise specific primer for the Chelonian that tortoise is found with other 11 kinds in Chinese field and corresponding application method.
Background technology
Red ear tortoise Trachemys scripta elegans are most dangerous one of 100 invasive species in the world, this kind of mesh
It is preceding successfully to be invaded in areas such as Europe, Africa and Asia, it does great damage to invasion ground ecological environment, and to social economy
It constitutes a serious threat with human health.Red ear tortoise is directed initially into China the 1980s, in recent years in China
Field is widely distributed, and potential ecological hazard is extremely serious, but for the quick knowledge technology of red ear tortoise invasion danger and prevention and control skill
Art system is not yet established, prevention and control scarce capacity of meeting an urgent need.In terms of the Field identification of red ear tortoise, common method be field investigation and
Largely there is time-consuming and laborious and inaccuracy in collection of specimens or evidence obtaining of taking pictures, these methods, because of red ear tortoise
It is sensitive to people, it is usually not easy directly to be found in water body, this just constrains the distribution of red ear tortoise field and quickly identifies opening for work
Exhibition.
With application of the Protocols in Molecular Biology in terms of species Field identification, environment DNA is utilized in recent years
The technology of the field distribution of (Environmental DNA) investigation animal and population density is continued to develop, which can pass through
The presence or absence of certain species specific DNA fragment is detected in environmental sample to judge that the species whether there is, and can be according to environment DNA
Concentration speculates animal population density, and this method can be in not collect specimen or prediction species point under conditions of find animal carcasses
Cloth and its density are a kind of efficient animal field investigation monitoring technology.Red ear tortoise inhabits fresh water environment, is practised for its life
Property, sample collection is mainly fresh water environment (river, pond and lake etc.), and also there is some other terrapin in Chinese field simultaneously
Class exists, therefore environment DNA may be being mixed for a variety of Chelonian DNA.Therefore, a kind of red ear tortoise specific primer use is developed
It is very necessary in environment DNA identification technology.
The specificity of primer is considered in design object species primer, while when expanding unknown template according to target fragment
The presence or absence of band distinguishes targeted species and other types, this technology is also referred to as Species specific PCR technology (species-
Specific PCR, ssPCR).Usually there are the genes such as COI, D-loop and Cyt b for the gene in Species specific PCR technology.
Mitochondrial DNA is not because its cyclic character is degradable, and copy number is more than core DNA, is widely used in environment DNA technology.Its
In, there is hypersensitivity, it is both in current species identification using wide when COI genes are expanded as target gene by special primer
One of species molecule label is commonly used in one of general molecular labeling and environment DNA technology.
Invention content
Present invention aims at：Provide a kind of red ear tortoise specificity that can quickly distinguish red ear tortoise with other 11 kinds of Chelonians
Primer and corresponding differentiating method.
In order to achieve the above-mentioned object of the invention, the present invention is adopted using the COI gene orders in red ear tortoise mitochondrial DNA as template
Primer mispairing, dimer and hairpin structure are checked with 5 softwares of Primer, devise red ear tortoise specific primer, nucleotides sequence
Row such as SEQ ID NO:Shown in 1~2.
The red ear tortoise specific primer of the present invention can be used for quickly distinguishing red ear tortoise and is found in Chinese field with other 11 kinds
Chelonian.
The described other 11 kinds Chelonians being found in Chinese field include (Chinese name and Latin literary fame)：Tortoise Mauremys
Reevesii, flower tortoise Mauremys sinensis, Mauremys mutica Mauremys mutica, peace cloth Cuora Cuora
Amboinensis, big fish tortoise Macroclemys temminckii, snapper turtle Chelydra serpentina, Platysternon megacephalum Gray
Platysternon megacephalum, Shelled Turtle Trionyx Sinensis Pelodiscus sinensis, Palea steindachneri Palea
Steindachneri, Asia soft-shelled turtle Amyda cartilaginea, Florida soft-shelled turtle Apaloneferox.
The present invention also provides a kind of methods for quickly distinguishing red ear tortoise and other 11 kinds of Chelonians, are that red ear tortoise is special
Property primer reacts for PCR and carries out electrophoresis detection, by the presence or absence of gained purpose band, quickly distinguishes red ear tortoise and other 11
Kind Chelonian.
Preferably, the reaction system of the PCR is：25 μ L, 2.0 μ L, 2 × Taq PCR Master Mix of template DNA
12.5 μ L, red ear tortoise specific primer each 1.0 μ L, ddH2O 8.5μL。
Preferably, the reaction condition of the PCR is：①94℃4min；2. 94 DEG C of 30s, 57 DEG C of 30s and 72 DEG C of 30s, totally 35
A cycle；3. 72 DEG C of extension 10min.
Preferably, the deposition condition is 140V, 35min.
Compared with the existing technology, the present invention has the advantages that：
Present invention could apply in the field investigation on distribution of red ear tortoise, since the distribution of red ear tortoise is relatively broad,
The problems such as work of field investigation takes time and effort uses using the COI gene orders in red ear tortoise mitochondrial DNA as template
5 softwares of Primer check primer mispairing, dimer and hairpin structure, devise red ear tortoise specific primer, can distinguish red ear
Tortoise and other 11 kinds of Chelonians common in Chinese Waters, and the effective molecular detecting method for distinguishing red ear tortoise is established, it is external
Quickly identification provides effective means to the distribution of invasive species red ear tortoise field, and requirement of its process to laboratory apparatus and consumptive material is not
Height, it is easy to utilize.
Description of the drawings
Fig. 1 is the electrophoresis result of the embodiment of the present invention 2；Wherein, M：2000bp DNAMarker；Swimming lane 1：Red ear tortoise
Trachemys scripta elegans；Swimming lane 2：Flower tortoise Mauremys sinensis；Swimming lane 3：Mauremys mutica
Mauremys mutica；Swimming lane 4：Snapper turtle Chelydra serpentina；Swimming lane 5：Pacify cloth Cuora Cuora
amboinensis；Swimming lane 6：Palea steindachneri Palea steindachneri；Swimming lane 7：Florida soft-shelled turtle Apaloneferox；Swimming lane
8：Asia soft-shelled turtle Amyda cartilaginea；Swimming lane 9：Shelled Turtle Trionyx Sinensis Pelodiscus sinensis；Swimming lane 10：Big fish tortoise
Macroclemys temminckii；Swimming lane 11：Platysternon megacephalum Gray Platysternon megacephalum；Swimming lane 12：Tortoise
Mauremys reevesii。
Specific implementation mode
In order to make the purpose of the present invention, technical solution and advantageous effects be more clear, with reference to embodiments, to this
Invention is further elaborated.It should be understood that embodiment described in this specification is just for the sake of this hair of explanation
It is bright, be not intended to limit the present invention, parameter, ratio of embodiment etc. can adaptation to local conditions make a choice and substance had no to result
It influences.
Embodiment 1
Using COI gene orders in red ear tortoise mitochondrial DNA (mtDNA) as template, check that primer is wrong using Primer5 softwares
Match, dimer and hairpin structure, designs red ear tortoise COI gene primers.It can be by red ear tortoise and other 11 kinds of tortoises by screening acquisition
The unique identification primer 1 that class distinguishes is right, and downstream sequence is thereon：
5'→3'GTTGGGATAGACGTAGACACC；
5'→3'CTCCGATAGTAAAGAGGAAGATG。
Embodiment 2
With the primer of the gained of embodiment 1 under the reaction system of the PCR of standard and reaction condition having by purpose band
Red ear tortoise and other 11 kinds of Chelonians are quickly distinguished in the difference of nothing.
PCR reaction systems be 25 μ L, template DNA 12.5 μ L of 2.0 μ L (about 100ng), 2 × Taq PCR Master Mix,
Upstream and downstream primer (100nmol/L) each 1.0 μ L, ddH2O 8.5μL.Reaction condition is 94 DEG C of 4min；94 DEG C of 30s, 57 DEG C of 30s,
72 DEG C of 30s, 35 cycles；72 DEG C of extension 10min.4 DEG C of preservations.Deposition condition is 140V, 35min.
Electrophoresis result refers to Fig. 1, it can be seen that the specific primer of 1 gained of embodiment can having by purpose band
Without distinguish red ear tortoise and flower tortoise, Mauremys mutica, snapper turtle, peace cloth Cuora, Palea steindachneri, Florida soft-shelled turtle, Asia soft-shelled turtle, in
Magnificent soft-shelled turtle, big fish tortoise, Platysternon megacephalum Gray and tortoise.
The announcement and guidance of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out change and modification appropriate.Therefore, the invention is not limited in specific implementation modes disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In used some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
Sequence table
<110>Guangdong Province's living resources Applied Research Laboratory
<120>A kind of red ear tortoise specific primer and its application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gttgggatag acgtagacac c 21
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctccgatagt aaagaggaag atg 23
Claims (8)
1. a kind of red ear tortoise specific primer, which is characterized in that its nucleotide sequence such as SEQ ID NO:Shown in 1~2.
2. red ear tortoise specific primer described in claim 1 is found with other 11 kinds in Chinese field quickly distinguishing red ear tortoise
Chelonian in application.
3. application according to claim 21, which is characterized in that other 11 kinds of Chelonians being found in Chinese field
Including：Tortoise Mauremys reevesii, flower tortoise Mauremys sinensis, Mauremys mutica Mauremys mutica, peace
Cloth Cuora Cuora amboinensis, big fish tortoise Macroclemys temminckii, snapper turtle Chelydra
Serpentina, Platysternon megacephalum Gray Platysternon megacephalum, Shelled Turtle Trionyx Sinensis Pelodiscus sinensis, Palea steindachneri
Palea steindachneri, Asia soft-shelled turtle Amyda cartilaginea, Florida soft-shelled turtle Apaloneferox.
4. a kind of method for quickly distinguishing red ear tortoise and other 11 kinds of Chelonians, which is characterized in that use red ear tortoise specific primer
Electrophoresis detection is reacted and carried out in PCR, by the presence or absence of gained purpose band, quickly distinguishes red ear tortoise and other 11 kinds of Chelonians；
The nucleotide sequence such as SEQ ID NO of the red ear tortoise specific primer:Shown in 1~2.
5. the method according to claim 4 for quickly distinguishing red ear tortoise and other 11 kinds of Chelonians, which is characterized in that described
The reaction system of PCR is：12.5 μ L of 25 μ L, 2.0 μ L, 2 × Taq PCR Master Mix of template DNA, red ear tortoise specificity are drawn
Object each 1.0 μ L, ddH2O 8.5μL。
6. the method according to claim 4 for quickly distinguishing red ear tortoise and other 11 kinds of Chelonians, which is characterized in that described
The reaction condition of PCR is：①94℃4min；2. 94 DEG C of 30s, 57 DEG C of 30s and 72 DEG C of 30s, totally 35 recycle；3. 72 DEG C of extensions
10min。
7. the method according to claim 4 for quickly distinguishing red ear tortoise and other 11 kinds of Chelonians, which is characterized in that the electricity
Swimming condition is 140V, 35min.
8. the method according to claim 4 for quickly distinguishing red ear tortoise and other 11 kinds of Chelonians, which is characterized in that it is described its
Its 11 kinds of Chelonian includes：Tortoise Mauremys reevesii, flower tortoise Mauremys sinensis, Mauremys mutica Mauremys
Mutica, peace cloth Cuora Cuora amboinensis, big fish tortoise Macroclemys temminckii, snapper turtle
Chelydra serpentina, Platysternon megacephalum Gray Platysternon megacephalum, Shelled Turtle Trionyx Sinensis Pelodiscus
Sinensis, Palea steindachneri Palea steindachneri, Asia soft-shelled turtle Amyda cartilaginea, Florida soft-shelled turtle Apalone
ferox。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810190950.3A CN108315434A (en) | 2018-03-08 | 2018-03-08 | A kind of red ear tortoise specific primer and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810190950.3A CN108315434A (en) | 2018-03-08 | 2018-03-08 | A kind of red ear tortoise specific primer and its application |
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| CN108315434A true CN108315434A (en) | 2018-07-24 |
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| CN201810190950.3A Pending CN108315434A (en) | 2018-03-08 | 2018-03-08 | A kind of red ear tortoise specific primer and its application |
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| CN110564868A (en) * | 2019-10-22 | 2019-12-13 | 生态环境部南京环境科学研究所 | red-eared turtle detection method and PCR primer for detecting red-eared turtles |
| CN110616271A (en) * | 2019-10-12 | 2019-12-27 | 生态环境部南京环境科学研究所 | Specific primer for quantitatively detecting biomass of red-eared turtle in environment and detection method and application thereof |
| CN116064848A (en) * | 2023-02-02 | 2023-05-05 | 生态环境部南京环境科学研究所 | Primer probe combination for quantitatively monitoring spatial and temporal distribution of DNA in red ear tortoise environment and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110616271A (en) * | 2019-10-12 | 2019-12-27 | 生态环境部南京环境科学研究所 | Specific primer for quantitatively detecting biomass of red-eared turtle in environment and detection method and application thereof |
| CN110616271B (en) * | 2019-10-12 | 2022-05-03 | 生态环境部南京环境科学研究所 | Specific primer for quantitatively detecting biomass of red-eared turtle in environment and detection method and application thereof |
| CN110564868A (en) * | 2019-10-22 | 2019-12-13 | 生态环境部南京环境科学研究所 | red-eared turtle detection method and PCR primer for detecting red-eared turtles |
| CN110564868B (en) * | 2019-10-22 | 2022-11-29 | 生态环境部南京环境科学研究所 | Red-eared turtle detection method and PCR primer for detecting red-eared turtles |
| CN116064848A (en) * | 2023-02-02 | 2023-05-05 | 生态环境部南京环境科学研究所 | Primer probe combination for quantitatively monitoring spatial and temporal distribution of DNA in red ear tortoise environment and application thereof |
| CN116064848B (en) * | 2023-02-02 | 2023-09-12 | 生态环境部南京环境科学研究所 | Primer probe combination for quantitatively monitoring spatial and temporal distribution of DNA in red ear tortoise environment and application thereof |
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Address after: 510260 No. 105 West Xingang Road, Guangzhou, Guangdong, Haizhuqu District Applicant after: Institute of zoology, Guangdong Academy of Sciences Address before: 510260 No. 105 West Xingang Road, Guangzhou, Guangdong, Haizhuqu District Applicant before: Guangdong Institute of Applied Biological Resources |
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Application publication date: 20180724 |