CN106978493A - A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid - Google Patents
A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid Download PDFInfo
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- CN106978493A CN106978493A CN201710254086.4A CN201710254086A CN106978493A CN 106978493 A CN106978493 A CN 106978493A CN 201710254086 A CN201710254086 A CN 201710254086A CN 106978493 A CN106978493 A CN 106978493A
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- tea geometrid
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- 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
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- 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]
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Abstract
A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid, belong to technical field of molecular biology.The present invention is using insect tissue as template, using special primer, after PCR is expanded, and digestion is carried out to its product using specific restriction enzyme.According to tea geometrid and the otherness of grey tea geometrid chondriogen specific site, tea geometrid and grey tea geometrid are distinguished by the band number of agarose electrophoresis：Tea geometrid does not haveFbaIRestriction enzyme site, the electrophoresis result of its digestion products is a band；Exist in grey tea geometrid sequenceFbaIRestriction enzyme site, the electrophoresis result of its digestion products is two bands.The present invention have it is simple to operate, be widely used, accuracy high and the features such as fast and cheap speed.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of Rapid identification tea geometrid is drawn with grey tea geometrid
Thing and its authentication method.
Background technology
Tea geometrid and grey tea geometrid are the important pests in China tea place.They belong to Lepidoptera, Geometridae, angstrom geometrid moth
Category, affiliation is extremely near.Both forms are very much like, generation area and time-interleaving, with identical host plant.In production
It is easy to obscure, disturbs the accuracy of agricultural pestses prediction.
Find according to the study, the ovum of tea geometrid and grey tea geometrid, larva, the similitude of pupa and adult reach human eye almost
The degree that can not be distinguished.Based on extremely near affiliation between the two, they often by scientist be used to probing into insect evolution and
The reason for species are formed.But how to go to distinguish tea geometrid and grey tea geometrid is the problem for perplexing entomologist always.Therefore, open
The authentication method for sending out quick and precisely tea geometrid and grey tea geometrid a kind of has very important meaning to production and scientific research.
The content of the invention
The problem of existing for prior art, it is an object of the invention to design to provide a kind of Rapid identification tea geometrid and ash
The primer of tea geometrid and its technical scheme of authentication method.
A kind of described Rapid identification tea geometrid and the primer of grey tea geometrid, it is characterised in that including primer Primer-F and
Primer-R, the primer Primer-F nucleotide sequence are as shown in SEQ ID No.1, the nucleosides of the primer Primer-R
Acid sequence is as shown in SEQ ID No.2.
A kind of described Rapid identification tea geometrid and the method for grey tea geometrid, it is characterised in that comprise the following steps：
1）Template is organized as with grey tea geometrid with tea geometrid, performing PCR amplification is entered using primer Primer-F and Primer-R, it is described
Primer Primer-F nucleotide sequence is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ of the primer Primer-R
Shown in ID No.2；
2）Digestion is carried out using restriction enzyme to amplified production；
3）Electrophoretic process is carried out to digestion products, tea geometrid and grey tea geometrid are distinguished by the band number of agarose electrophoresis,
It is tea geometrid if electrophoretic band is one, two are grey tea geometrid.
A kind of described Rapid identification tea geometrid and the method for grey tea geometrid, it is characterised in that the restriction enzyme isFbaI。
The present invention is according to tea geometrid and the otherness of grey tea geometrid chondriogen specific site（FbaIRestriction enzyme site
The presence or absence of " TGATCA "）, tea geometrid and grey tea geometrid are distinguished by the band number of agarose electrophoresis：Tea geometrid does not haveFbaI
Restriction enzyme site, the electrophoresis result of its digestion products is a band；Exist in grey tea geometrid sequenceFbaIRestriction enzyme site, its digestion production
The electrophoresis result of thing is two bands.
The invention has the advantages that：
1. it is simple to operate.The PCR and enzyme incision technology that the present invention is used are traditional Protocols in Molecular Biology, technology maturation, behaviour
Make simple.
It 2. is widely used.The present invention can be for identification any period and the tea geometrid and grey tea geometrid of worm state.
3. accuracy is high.It is mutated to distinguish tea geometrid and grey tea geometrid according to the restriction enzyme site on DNA sequence dna, can be reached
100% accuracy rate.
4. speed is fast and cheap.The present invention distinguishes tea using insect tissue as pcr template, and with endonuclease reaction
Looper and grey tea geometrid, can obtain result within 4 hours.DNA cloning and the method for sequencing than before, save DNA
Extract and expense and time needed for DNA sequencing.
Brief description of the drawings
Fig. 1 is the identification of the tea geometrid and grey tea geometrid of indoor feeding in the embodiment of the present invention 1；
Fig. 2 is the identification of the tea geometrid and grey tea geometrid of field collection in the embodiment of the present invention 2.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual（Sambrook J & Russell DW,
Molecular cloning: a laboratory manual,2001）, or according to the condition of manufacturer's specification suggestion.
Embodiment 1：Tea geometrid and grey tea geometrid identification that indoors artificial is raised
1）Synthesize following primer：
Primer-F：5’-AAATGTTGGTATAAAATTGGGTCTCC-3’（SEQ ID No.1）
Primer-R：5’-AATACTATTGTAACCGCTCATGCTT-3’ （SEQ ID No.2）.
2）Tea geometrid and grey adults of ectropis obliqua is taken to distinguish 6 and 4 respectively.
3）A fritter tissues of sample foot position are taken, tissue size is：Diameter 1mm, thickness 0.7mm.
4）PCR is expanded, and reaction system is as follows：
Response procedures are：98 DEG C of denaturation 2min；Carry out 35 cyclic amplification reactions（98 DEG C of denaturation 10s, 55 DEG C of anneal 15s, 68 DEG C
Extend 40s.）；12 DEG C of holdings.
5）Reaction takes out PCR reaction tubes after terminating, and carries out endonuclease reaction, and reaction system is：
Reaction condition is：37 DEG C of reaction 1h.
6）Detect that reaction product, electrophoresis are imaged after terminating in gel imager with 1.5% agarose gel electrophoresis.Mirror
Determine result to show, tea geometrid has an electrophoretic band, and grey tea geometrid there are two（Fig. 1）, and result known to qualification result
Unanimously, illustrate the method rate of accuracy reached to 100%.
Meanwhile, after amplified production is sequenced, the tea geometrid DNA sequence dna different from grey tea geometrid acquisition, the DNA sequences of tea geometrid
Row are as shown in SEQ ID No.3, and the DNA sequence dna of grey tea geometrid is as shown in SEQ ID No.4.
Embodiment 2：The tea geometrid of field collection and the identification of grey tea geometrid
1）Synthesize following primer：
Primer-F：5’-AAATGTTGGTATAAAATTGGGTCTCC-3’ （SEQ ID No.1）
Primer-R：5’-AATACTATTGTAACCGCTCATGCTT-3’ （SEQ ID No.2）.
2）Entomologize larva in Hangzhou West Lake area, Shaoxing and Hangzhou Yuhang District tea place respectively（It is unknown be tea geometrid also
It is grey tea geometrid）.
3）A fritter tissues of sample stern foot position are taken, tissue size is：Diameter 1mm, thickness 0.7mm.
4）PCR is expanded, and reaction system is as follows：
Response procedures are：98 DEG C of denaturation 2min；Carry out 35 cyclic amplification reactions（98 DEG C of denaturation 10s, 55 DEG C of anneal 15s, 68 DEG C
Extend 40s.）；12 DEG C of holdings.
5）Reaction takes out PCR reaction tubes after terminating, and carries out endonuclease reaction, and reaction system is：
Reaction condition is：37 DEG C of reaction 1h.
6）Detect that reaction product, electrophoresis are imaged after terminating in gel imager with 1.5% agarose gel electrophoresis.From
Fig. 2 qualification result shows, Hangzhou West Lake area（Fig. 2A）And Yuhang District（Fig. 2 C）Sample in existing tea geometrid also have grey tea chi
Earwig, and Shaoxing Yu Chacun（Fig. 2 B）Sample there was only grey tea geometrid.
SEQUENCE LISTING
<110>Tea Inst., Chinese Academy of Agricultural Sciences
<120>A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid
<130> 1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 1
aaatgttggt ataaaattgg gtctcc 26
<210> 2
<211> 25
<212> DNA
<213>It is artificial synthesized
<400> 2
aatactattg taaccgctca tgctt 25
<210> 3
<211> 539
<212> DNA
<213>Tea geometrid
<400> 3
aaatgttggt ataaaattgg gtctcctcct ccagcagggt caaaaaaaga tgtatttaaa 60
tttcgatcag ttagtaatat tgtaatagcc ccagctaaaa ctggtaaaga taaaagtaat 120
aaaaatgctg taattccaac agctcaaaca aataaaggta tttggtcaaa tgataaatta 180
tttaatcgta tattaataat tgttgtaata aagttaatag ctccaagaat tgaagaaata 240
ccagctaaat gtagtgaaaa aattgctaaa tctacagatc ttccaccatg agcaatatta 300
gaggataaag gagggtaaac tgttcatcct gttcctgctc cattttctac aattcttcta 360
gaaattagta aagtaagaga tgggggtaat aatcaaaatc ttatattatt tattcgtggg 420
aaagctatat caggggctcc taatattaaa ggtactaatc agtttccaaa tcctccaatt 480
ataataggta ttactataaa aaaaattata ataaaagcat gagcagttac aatagtatt 539
<210> 4
<211> 539
<212> DNA
<213>Grey tea geometrid
<400> 4
aaatgttgat ataaaattgg atctcctccc ccagctgggt caaaaaaaga tgtatttaaa 60
tttcgatcag ttaataatat tgtaatagcc ccagctaaaa ctggtaaaga taagagtaat 120
aagaatgctg taattccaac agctcaaaca aataaaggta tctgatcaaa tgataaatta 180
tttaatcgta tattaataat tgttgtaata aaattaatag ccccaagaat tgaggaaata 240
ccagctaaat gtaatgaaaa aattgctaaa tctactgatc ttccaccgtg agcaatatta 300
gaagataaag gaggataaac tgttcatcct gttcctgctc cattttctac aattcttcta 360
gaaattaata aagtaagaga tgggggtaat agtcaaaatc ttatattatt tattcgtggg 420
aaagctatat caggagctcc taatattaaa ggtactaatc aatttccaaa tcctccgatt 480
ataattggta ttactataaa aaaaattata ataaaagcat gagcggttac aatagtatt 539
Claims (3)
1. a kind of Rapid identification tea geometrid and the primer of grey tea geometrid, it is characterised in that including primer Primer-F and Primer-
R, the primer Primer-F nucleotide sequence are as shown in SEQ ID No.1, the nucleotide sequence of the primer Primer-R
As shown in SEQ ID No.2.
2. a kind of Rapid identification tea geometrid and the method for grey tea geometrid, it is characterised in that comprise the following steps：
1）Template is organized as with grey tea geometrid with tea geometrid, performing PCR amplification is entered using primer Primer-F and Primer-R, it is described
Primer Primer-F nucleotide sequence is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ of the primer Primer-R
Shown in ID No.2；
2）Digestion is carried out using restriction enzyme to amplified production；
3）Electrophoretic process is carried out to digestion products, tea geometrid and grey tea geometrid are distinguished by the band number of agarose electrophoresis,
It is tea geometrid if electrophoretic band is one, two are grey tea geometrid.
3. a kind of Rapid identification tea geometrid as claimed in claim 2 and the method for grey tea geometrid, it is characterised in that the limitation
Property restriction endonuclease isFbaI。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710254086.4A CN106978493A (en) | 2017-04-18 | 2017-04-18 | A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710254086.4A CN106978493A (en) | 2017-04-18 | 2017-04-18 | A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid |
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| CN106978493A true CN106978493A (en) | 2017-07-25 |
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ID=59345167
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|---|---|---|---|
| CN201710254086.4A Pending CN106978493A (en) | 2017-04-18 | 2017-04-18 | A kind of Rapid identification tea geometrid and the primer and its authentication method of grey tea geometrid |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342492A (en) * | 2018-05-07 | 2018-07-31 | 山西农业大学 | A kind of primer and its discrimination method of quick discriminating Candle-sticks stinkbugs and eating attraction, Dolycoris baccarum |
| CN109971866A (en) * | 2019-04-18 | 2019-07-05 | 浙江大学 | A kind of primer, kit and method for identifying grey ectropis obliqua |
| CN111411160A (en) * | 2020-05-21 | 2020-07-14 | 四川省农业科学院植物保护研究所 | Method for identifying tea geometrid by utilizing mitochondrial molecular marker and application thereof |
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|---|---|---|---|---|
| CN101831508A (en) * | 2010-05-27 | 2010-09-15 | 中国农业科学院茶叶研究所 | Quantitative detection method of tea-geometrid-type polyhedrosis viruses |
| CN102344919A (en) * | 2010-08-04 | 2012-02-08 | 中国农业大学 | Direct PCR (Polymerase Chain Reaction) method without extracting animal tissue DNA (Deoxyribonucleic Acid) |
| CN103583286A (en) * | 2013-10-28 | 2014-02-19 | 中国农业科学院茶叶研究所 | Genetic control method for tea geometrids |
| CN105145515A (en) * | 2015-08-26 | 2015-12-16 | 中国农业科学院茶叶研究所 | Method for controlling and monitoring tea geometrids using volatile attractant |
| CN105685041A (en) * | 2016-01-13 | 2016-06-22 | 中国农业科学院茶叶研究所 | Application of pheromone composition to controlling ectropis grisescens |
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2017
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101831508A (en) * | 2010-05-27 | 2010-09-15 | 中国农业科学院茶叶研究所 | Quantitative detection method of tea-geometrid-type polyhedrosis viruses |
| CN102344919A (en) * | 2010-08-04 | 2012-02-08 | 中国农业大学 | Direct PCR (Polymerase Chain Reaction) method without extracting animal tissue DNA (Deoxyribonucleic Acid) |
| CN103583286A (en) * | 2013-10-28 | 2014-02-19 | 中国农业科学院茶叶研究所 | Genetic control method for tea geometrids |
| CN105145515A (en) * | 2015-08-26 | 2015-12-16 | 中国农业科学院茶叶研究所 | Method for controlling and monitoring tea geometrids using volatile attractant |
| CN105685041A (en) * | 2016-01-13 | 2016-06-22 | 中国农业科学院茶叶研究所 | Application of pheromone composition to controlling ectropis grisescens |
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| Title |
|---|
| ZHANG GH等: "Asymmetrical reproductive interference between two sibling species of tea looper: Ectropis grisescens and Ectropis obliqua", 《BULL ENTOMOL RES》 * |
| ZHANG GH等: "Detecting deep divergence in seventeen populations of tea geometrid (Ectropis obliqua Prout) in China by COI mtDNA and cross-breeding", 《PLOS ONE》 * |
| 姜楠等: "我国华东地区两种茶尺蠖的形态和分子鉴定", 《应用昆虫学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342492A (en) * | 2018-05-07 | 2018-07-31 | 山西农业大学 | A kind of primer and its discrimination method of quick discriminating Candle-sticks stinkbugs and eating attraction, Dolycoris baccarum |
| CN108342492B (en) * | 2018-05-07 | 2021-02-02 | 山西农业大学 | Primer for rapidly identifying lugworm bugs, tea-wing bugs and stinkbug and identification method thereof |
| CN109971866A (en) * | 2019-04-18 | 2019-07-05 | 浙江大学 | A kind of primer, kit and method for identifying grey ectropis obliqua |
| CN111411160A (en) * | 2020-05-21 | 2020-07-14 | 四川省农业科学院植物保护研究所 | Method for identifying tea geometrid by utilizing mitochondrial molecular marker and application thereof |
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Application publication date: 20170725 |