CN109182547A - It is a kind of for identifying the primer pair and its application of grape flower wing steinernema - Google Patents
It is a kind of for identifying the primer pair and its application of grape flower wing steinernema Download PDFInfo
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- CN109182547A CN109182547A CN201811228694.9A CN201811228694A CN109182547A CN 109182547 A CN109182547 A CN 109182547A CN 201811228694 A CN201811228694 A CN 201811228694A CN 109182547 A CN109182547 A CN 109182547A
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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
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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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- 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
The present invention provides a kind of primer pair for identifying grape flower wing steinernema, and upstream primer nucleotide sequences are as shown in SEQ ID NO:1, it may be assumed that 5 '-CAAAATAGAGCCTCCCCT-3 '；Primer nucleotide sequences are as shown in SEQ ID NO:2 downstream, it may be assumed that 5 '-AAACGACCTGGATTAGCAT-3 '.The present invention provides the applications of above-mentioned primer pair.The present invention provides the specific primer pair of grape flower wing steinernema, and identifies grape flower wing steinernema using the primer pair, and this method has the characteristics that quick, qualification result is accurate, easily operated, high sensitivity and specificity are strong.
Description
Technical field
The invention belongs to molecular identification technical fields, and in particular to one kind is for identifying quarantine pest grape flower wing rouleau
The primer pair of moth, and the method for identifying molecules using the primer pair grape flower wing steinernema.
Background technique
Grape flower wing steinernema (Lobesia botrana) belongs to Lepidoptera (Lepidoptera), Tortricidae
(Tortricidae), flower wing steinernema category (Lobesia), for the worldwide evil for seriously endangering diversified economy plant flowers and fruit
Worm includes that our country is listed as important inward plant quarantine harmful insects in world many countries.The worm source area is that meaning is big
Benefit has reached all Europe, Africa northern and the western, Middle East in Asia and the Far East Area and Some Areas of USA, at present I
There are no the relevant reports that this kind occurs for state.
A 1 year generation generation at most generation of the worm, the factors such as specific algebra and photoperiod and temperature are related.Its first brood of larvae
Grape bud is endangered, larva endangers grape fruit after the second generation, generates direct economic loss；After grape fruit is aggrieved, wound
It is easy to be infected by grape grey mould, causes gray mold popular, cause more huge economic loss.Grape flower wing steinernema adult is general
Start the activities such as mated, laid eggs after sunset dusk.In addition, the host plant of grape flower wing steinernema is also in addition to grape
There are 40 various plants such as apple, Lee, persimmon, cherry, Kiwi berry, pomegranate, olive etc., nectarine, once incoming China's fruit
Important producing region, producing and selling, the outlet for more China's fruit that are bound to cause significant impact, to seriously threaten the peace of China's fruit
Full production, has great importance so carrying out prevention and control to it with epidemic situation intervention.
Currently, the identification for grape flower wing steinernema uses traditional Morphological Identification, in the pest of Lepidoptera Tortricidae
In, grape flower wing steinernema has morphological feature below: adult figure belongs to middle-size and small-size moth class insect, the long 4.5- of body
5.5mm, wing expanse 10-13mm, adult size are related to larval feeding.Feeler is Filamentous, light brown.Head is cream-colored, and the crown
Fasciation grey fine strip shape scopular；Back give birth to apparent brown back feathering (Niu Chun respect, and 2013. quarantine nocuousness are raw
Object-grape flower wing steinernema [J] inspection and quarantine academic periodical).Nearby edge has recessed part to push up also by part outstanding to fore wing
Cornicult goes out, and edge has fine, soft fur, and full wing has three pieces significantly by black and the light brown mottling figure line formed, and edge is white
Color.When adult is static, it is in hang mitriform that two wings, which merge,.The difference of female male imago be mainly external genital organs (Li Junfeng etc., 2017.
Worldwide pest grape flower wing steinernema invades risk analysis [J] the bio-safety journal in China).
Morphological Identification method the professional knowledge of assessor and the sample integrity of grape flower wing steinernema are required to it is higher, and
And heart-eating worm class is easily obscured with form allied species, causes the identification of grape flower wing steinernema time-consuming and laborious, and accuracy and repetition
Property is not high.Traditional morphological method is difficult to meet small to grape flower wing during port quarantine and fruit and seedling allocation and transportation
Volume moth quickly identifies the actual demand with identification, especially when the sample of intercepting and capturing is larva or residuum, and grape flower wing steinernema
Quick and precisely identification be the important prerequisite for effectively preventing it that diffusion is propagated further.
Summary of the invention
Identification method for grape flower wing steinernema is Morphological Identification, to the professional knowledge of sample quality and appraiser
It is required that all very high, applicability is low, and identification difficulty is big, and the above problem that qualification result accuracy is low, and the present invention provides grape flower
The specific primer pair of wing steinernema, and grape flower wing steinernema is identified using the primer pair, this method has quick, identification
As a result the features such as accurate, easily operated, high sensitivity and specificity are strong.
Realization that the present invention adopts the following technical solutions:
It is a kind of identify grape flower wing steinernema primer pair, upstream primer nucleotide sequences as shown in SEQ ID NO:1,
That is: 5 '-CAAAATAGAGCCTCCCCT-3 '；Primer nucleotide sequences are as shown in SEQ ID NO:2 downstream, it may be assumed that 5 '-
AAACGACCTGGATTAGCAT-3’。
Firstly, finding grape flower wing steinernema on NCBI and reported 10 Lepidopterous Tortricidae is close with its form
Like the mitochondrial COII gene sequence of kind；
Secondly the COII gene order of grape flower wing steinernema and 10 kinds of form allied species is carried out pair using MEGA6.0
Than so that it is determined that the distinctive COII genetic fragment of grape flower wing steinernema out；
Finally grape flower wing rouleau is designed for the distinctive COII genetic fragment of grape flower wing steinernema using Primier5
Moth specific primer pair.
Designed primer pair grape flower wing steinernema population carries out PCR amplification, can get the specific piece of 508bp size
Section.
The present invention also provides a kind of kits for identifying grape flower wing steinernema, and it includes above-mentioned primer pairs.Further
Ground also includes PCR buffer, Taq archaeal dna polymerase, ddH20。
The present invention also provides a kind of methods with the primer pair identification grape flower wing steinernema, and this method is: extracting
The genomic DNA of sample to be identified utilizes the mitochondria in primer pair genomic DNA described in claim 1 as template
COII gene carries out PCR amplification, detects to amplified production, if there is the DNA band of 508bp, sample to be tested is grape
Flower wing steinernema.
In the above method, pcr amplification reaction condition are as follows: initial denaturation 3 minutes at 94 DEG C；Then 35 circulations, each circulation
It is denaturalized 30 seconds including 94 DEG C, 53 DEG C are annealed 30 seconds, 72 DEG C of extension 30s；Last 72 DEG C of extensions 5min.
The present invention also provides a kind of PCR reaction systems using primer pair identification grape flower wing steinernema comprising:
2 × PCR Master mix, 12.5 μ l, upstream and downstream contain 20~50ng to each 2 μ l (10mM) of primer, 1.5 μ l of DNA profiling
DNA, ddH2O supplies 25 μ l.
The present invention obtains the distinguished sequence of a COII gene of grape flower wing steinernema by the method for Molecular Identification, so
Afterwards by the comparison of gene order similarity, it can be achieved that grape flower wing steinernema is accurately and rapidly identified.(1) operation of the present invention
Simply, operator does not need the Morphological Identification knowledge for having profession；And traditional identification method is complicated for operation, and needs to have
Stronger Morphological Identification knowledge.(2) result of the present invention is accurate and reliable, the method for the present invention to pick up from Chile, Argentina, with color
Column, Italian grape flower wing steinernema carry out identification verifying, and reliability as a result, which has, adequately to be guaranteed.(3) present invention is real
It is strong with property, it can be used for the identification of grape flower wing rouleau moth larvae, pupa, adult, can reflect as long as obtaining any a part of polypide
It is fixed, to sample quality no requirement (NR), it can be used for the prediction of Field Pests, it can also be used to pass in and out port to Imported Fruits, nursery stock
Detection, the clearance time can not only be shortened, but also can reduce the working strength of quarantine functionary；And traditional identification method
And sample quality complete to sample is relatively high.(4) identify that the specific primer of grape flower wing steinernema can amplify grape
The mitochondrial COII gene of flower wing steinernema provides easy stable molecular labeling for the identification of grape flower wing steinernema, solves
The problem of grape flower wing steinernema is distinguished, it is anti-for the biology of grape flower wing steinernema from now on, population dynamic monitoring and synthesis
It controls and lays a good foundation.
Detailed description of the invention
Fig. 1 is grape flower wing steinernema primer pair acquisition Chile, Argentina, Israel, Italian four places certainly
The amplification of 12 parts of grape flower wing steinernema sample total DNAs, wherein M:DL2000marker；1-3 is acquisition Chilean Portugal certainly
Grape wing steinernema sample；4-6 is to acquire from Argentine grape flower wing steinernema sample；7-9 is to acquire from the Portugal of Israel
Grape wing steinernema sample；10-12 is grape flower wing steinernema sample of the acquisition from Italy.
Specific embodiment
Combined with specific embodiments below and attached drawing is described in further details the present invention.
Embodiment 1 is used to identify the specific PCR primers sequence design of grape flower wing steinernema
Firstly, finding grape flower wing steinernema on NCBI and reported 10 Lepidopterous Tortricidae is close with its form
Like the mitochondrial COII gene sequence of kind；
Secondly the COII gene order of grape flower wing steinernema and 10 kinds of form allied species is carried out pair using MEGA6.0
Than so that it is determined that the distinctive COII genetic fragment of grape flower wing steinernema out；
Finally grape flower wing rouleau is designed for the distinctive COII genetic fragment of grape flower wing steinernema using Primier5
Moth specific primer pair.
According to distinguished sequence design primer derived above, primer sequence are as follows:
Upstream primer nucleotide sequence is as described in SEQ ID NO:1, it may be assumed that 5 '-CAAAATAGAGCCTCCCCT-3 '；Under it
Primer nucleotide sequences are swum as described in SEQ ID NO:2, it may be assumed that 5 '-AAACGACCTGGATTAGCAT-3 '.
Embodiment 2PCR primer sequence is for identifying grape flower wing steinernema
1. the acquisition of insect sample
3-5 month in 2018 is in Chile, Argentina's acquisition grape flower wing rouleau moth larvae, Israel, acquisition Portugal of Italy
Grape wing rouleau moth larvae and adult are placed in dehydrated alcohol through the accurate Morphological Identification of expert, are stored in -20 DEG C.
1 present invention grape flower wing steinernema sample information collected of table
2. extracting genome DNA
By the sample of collection, diluted with the alcohol that concentration gradient is 75%, 50%, 30%, 10%, 0%, each concentration
2min；It is taken out from sterile water, the moisture on polypide is blotted with filter paper；Sample is placed in a sterile 1.5ml centrifuge tube；With
After grinding rod is fully ground, 100 μ lLB are added2With 20 μ l Proteinase K (ensuring tissue completely into centrifuge tube)；55
DEG C be incubated for about 3 hours until completely cracking；20 μ l RNase A are added in sample, are incubated at room temperature 2 minutes；12000rpm from
The heart 5 minutes, supernatant was shifted in a sterile centrifugation tube；500 μ lBB2 are added, are vortexed 5 seconds immediately, are incubated at room temperature 10 minutes；It will
Whole solution is added in centrifugal column, and 12000rpm is centrifuged 30 seconds, discards efflux；500 μ l solution C B2,12000rpm are added
Centrifugation 30 seconds, discards efflux, is repeated once；500 μ l solution W B2 (whether preoperation inspection is added dehydrated alcohol) are added,
12000rpm is centrifuged 30 seconds, is discarded efflux, is repeated once；12000rpm is centrifuged 2 minutes, completely removes remaining WB2；It will be from
Stem is placed in a clean centrifuge tube, the EB or deionized water (PH > 7.0) of 20 μ preheating is added in the center of column, room temperature is quiet
It sets one minute, 12000rpm is centrifuged one minute, eluted dna；Repetition afford more DNA, by the DNA being eluted out in-
20 DEG C of preservations.
Amplification, sequencing and the sequence analysis of 3.COII gene
(1) amplification of gene
Using genomic DNA obtained by previous step as template, the primer pair (upstream primer: 5 '-of the design of embodiment 1 is utilized
CAAAATAGAGCCTCCCCT-3 ', C2R: downstream primer: 5 '-AAACGACCTGGATTAGCAT-3 ') in genomic DNA
Mitochondrial COII gene carries out PCR amplification；
PCR reacts 25 μ l of total system, wherein 2 × PCR Master mix 12.5 μ l, positive/negative to each 2 μ l, DNA mould of primer
Plate 1.5 μ l, ddH2O supplies 25 μ l；
PCR reaction condition are as follows: initial denaturation 3 minutes at 94 DEG C；Then 35 circulations, each circulation include 94 DEG C of denaturation 30
Second, 53 DEG C are annealed 30 seconds, 72 DEG C of extension 30s；Last 72 DEG C of extensions 5min, reaction product are stored in 4 DEG C.
(2) sequence is analyzed
Appropriate pcr amplification product agarose electrophoresis testing result obtained in the previous step is taken to make after ethidium bromide staining
With 1% agarose electrophoresis, observed under ultraviolet lamp.Above-mentioned primer amplification clip size is 508bp, and obtained specificity is produced
Object sequencing, is compared, thus to identify grape flower wing steinernema according to the result of sequencing.
4. experimental result
The sequence that resulting COII gene is sequenced is similar with the sequence for the grape flower wing steinernema searched in NCBI
Degree is 100%, identifies that accuracy rate is 100%.
3 grape flower wing steinernema of embodiment and other allied species sequence alignments
1. downloading the COII gene sequence of reported Lepidoptera Tortricidae species approximate with grape flower wing steinernema in NCBI
Column.
2. the grape flower wing steinernema COII gene order being sequenced using primer amplified provided by the invention
It is compared with the COII gene order of other allied species by software CLUSTALX1.83.
3. experimental result
Pass through sequence alignment, it has been found that the grape flower wing that the primer amplified provided through the invention is sequenced
The grape flower wing steinernema COII gene order similarity downloaded on steinernema COII gene order and NCBI is 100%,
Remaining 10 species approximate with its and grape flower wing steinernema COII gene order similarity are 92% hereinafter, illustrate the present invention
Provided primer specificity is strong.
Sequence table 1 is the COII of grape flower wing steinernema sequence species approximate with the Lepidoptera Tortricidae 10 having been reported
Sequence alignment, wherein Ah represents the COII gene order of tea olethreutid (Adoxophyes honmai), Serial No.
DQ073916.1；Gd represents the COII gene order of Grapholita dimorphaKomai (Grapholita dimorpha), Serial No.
KJ671625.1；Af represents the COII gene order of AclerisfimbrianaMeyrick (Acleris fimbriana)；Ao represents applied perfume
The COII gene order of (Adoxophyes orana), Serial No. JX872403.1；Cp represents carpocapsa pononella (Cydia
Pomonella COII gene order), Serial No. JX407107.2；Rl represents thin tip steinernema (Rhyacionia
Leptotubula COII gene order), Serial No. JX028270.1；Gm represents oriental fruit months (Grapholita
Molesta COII gene order), Serial No. HQ392511.1；Sl represents Spilonota lechriaspis (Spilonota
Lechriaspis COII gene order), Serial No. HQ392511.1；Cl represents the big leaf roller of apple
The COII gene order of (Choristoneura longicellana), Serial No. HQ452340.1；It is small in fact that Rp represents Douglas fir
Roll up the COII gene order of moth (Retinia pseudotsugaicola), Serial No. KF498969.1；Lb represents grape flower
The COII gene order of wing steinernema (Lobesia botrana), the primer amplified which provides through the invention
Sequencing obtains.
The following contents is the grape flower wing steinernema obtained by GeneDoc software and its allied species COII gene order
It compares:
Embodiment described above is only that preferred embodiments of the present invention will be described, not to the scope of the present invention
It is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical solution of the present invention
The various changes and improvements made should all be fallen into the protection scope that claims of the present invention determines.
Sequence table
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of for identifying the primer pair and its application of grape flower wing steinernema
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caaaatagag cctcccct 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aaacgacctg gattagcat 19
Claims (5)
1. it is a kind of identify grape flower wing steinernema primer pair, upstream primer nucleotide sequences as shown in SEQ ID NO:1,
Downstream primer nucleotide sequence is as shown in SEQ ID NO:2.
2. a kind of kit for identifying grape flower wing steinernema, which is characterized in that include primer pair described in claim 1.
3. kit according to claim 2, which is characterized in that also comprising PCR buffer, Taq archaeal dna polymerase,
ddH20。
4. a kind of method with primer pair described in claim 1 identification grape flower wing steinernema, which is characterized in that extract wait reflect
The genomic DNA of random sample sheet utilizes the mitochondrial COII base in primer pair genomic DNA described in claim 1 as template
Because carrying out PCR amplification, amplified production is detected, if there is the DNA band of 508bp, sample to be tested is that grape flower wing is small
Roll up moth.
5. according to the method described in claim 4, it is characterized in that, pcr amplification reaction condition are as follows: initial denaturation 3 minutes at 94 DEG C；
Then 35 circulations, each circulation include 94 DEG C and are denaturalized 30 seconds, and 53 DEG C are annealed 30 seconds, 72 DEG C of extension 30s；Last 72 DEG C of extensions
5min。
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| CN201811228694.9A CN109182547B (en) | 2018-10-22 | 2018-10-22 | Primer pair for identifying grape flower wing diamond back moth and application thereof |
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| CN109182547B CN109182547B (en) | 2021-11-23 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882116A (en) * | 2014-01-22 | 2014-06-25 | 西北农林科技大学 | Molecular identification primer for four fruit tree core-eating insects and using method of molecular identification primer |
| CN104651508A (en) * | 2015-02-12 | 2015-05-27 | 珠海出入境检验检疫局检验检疫技术中心 | Fluorescent quantitative PCR molecule detection method of Coptotermestravians Haviland based on COII genes |
| CN105506103A (en) * | 2015-12-28 | 2016-04-20 | 天津中医药大学 | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof |
-
2018
- 2018-10-22 CN CN201811228694.9A patent/CN109182547B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882116A (en) * | 2014-01-22 | 2014-06-25 | 西北农林科技大学 | Molecular identification primer for four fruit tree core-eating insects and using method of molecular identification primer |
| CN104651508A (en) * | 2015-02-12 | 2015-05-27 | 珠海出入境检验检疫局检验检疫技术中心 | Fluorescent quantitative PCR molecule detection method of Coptotermestravians Haviland based on COII genes |
| CN105506103A (en) * | 2015-12-28 | 2016-04-20 | 天津中医药大学 | Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof |
Non-Patent Citations (2)
| Title |
|---|
| L. A. LEDEZMA等: "Diagnosis of Lobesia botrana (Lepidoptera: Tortricidae) Using Real-Time PCR", 《JOURNAL OF ECONOMIC ENTOMOLOGY》 * |
| MELISSA CLAIRE PIPER等: "Complete mitochondrial genome of the European Grapevine moth (EGVM) Lobesia botrana (Lepidoptera: Tortricidae)", 《MITOCHONDRIAL DNA THE JOURNAL OF DNA MAPPING, SEQUENCING, AND ANALYSIS》 * |
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