CN107164500A - Detect primer, kit and the detection method of Douglas fir large small moth - Google Patents
Detect primer, kit and the detection method of Douglas fir large small moth Download PDFInfo
- Publication number
- CN107164500A CN107164500A CN201710448505.8A CN201710448505A CN107164500A CN 107164500 A CN107164500 A CN 107164500A CN 201710448505 A CN201710448505 A CN 201710448505A CN 107164500 A CN107164500 A CN 107164500A
- Authority
- CN
- China
- Prior art keywords
- primer
- pcr
- large small
- douglas fir
- dna
- 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.)
- Granted
Links
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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (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 provides primer, kit and the detection method of detection Douglas fir large small moth.The detection method is nest-type PRC, and primer used is as shown in SEQ ID NO.1 and SEQ ID NO.2.Present invention also offers the kit of the detection Douglas fir large small moth including above-mentioned primer.The present invention detection method and its kit have the advantages that it is simple to operate, accurately and reliably, detection time it is short and specific good, Douglas fir large small moth can be made a distinction with other bark beetles, detect be limited to 20pg.
Description
Technical field
The present invention relates to biology field, more particularly, to the primer of detection Douglas fir large small moth, kit and
Detection method.
Background technology
Douglas fir large small moth (Dendroctonus pseudotsugae Hopkins.) is that the intercept and capture frequency is most in recent years
Many large small moths, have seriously endangered forest and timber resources.In recent years, taken in timber, woodwork and the wooden packing largely entered a country
The wood borers of band are extremely complex, common are longicorn, bark beetle, long moth, Ji Ding, weevil etc..Wherein, bark beetle is because of its epidemic situation complexity
In addition it is small (often less than 9mm) into polypide, cause its Rapid identification very difficult, current port can identify the deficiency planted
12%.According to statistics, the intercepting and capturing frequency of Douglas fir large small moth accounts for the 36.31% of the total frequency of large small moth intercepting and capturing, than the cloud for occupying second frequency
China fir large small moth will be higher by 29.3%.For effective prevention and control Exotic pests, first have to carry out it in the view of the scene of port
Accurately and quickly identify.How to intercept and capture with a definite target in view and judge Douglas fir large small moth epidemic situation, improve the specific aim of quarantine and have
Effect property, the production safety and ecological safety of the forestry of protection China, the Rapid identification work for solving Douglas fir large small moth seems particularly heavy
Will.
The research for being directed to Douglas fir large small moth both at home and abroad at present is less, and is concentrated mainly on adult morphology aspect, together
When both at home and abroad for Douglas fir large small moth identification mainly pass through adult morphological method, this method need specialty entomological taxonomy
Knowledge, higher to personnel requirement, the Douglas fir size moth state carried in timber, woodwork and the wooden packing largely entered a country is complicated,
Adult Senile Mouse is difficult to deal with, and requires that clearance speed is fast during port quarantine, it is impossible to raised ovum, larva to adult
Identify again afterwards.
DNA bar code technology is, the gene piece that is readily available moderate using biological one section of conservative generally having itself
Duan Zuowei standards, a kind of species identification means set up on modern advanced DNA cloning, sequencing and comparison technology basis.
Compared with traditional Morphological Identification, carry out species identification using DNA bar code and have the advantage that：Identification to species will no longer
Limited by species developmental condition, overcome ovum, larva, pupa etc. can not Direct Identification shortcoming；To the experience of assessor and specially
Industry background knowledge is substantially reduced, and reduces the interference of subjective judgement, and the identification of species is more accurate quick.
In recent years, species are identified using molecular engineering and phylogenetic relationship research has become one kind and quickly had
The method of effect, the wherein terminal sequence of chondriogen 5 ' have species Genetic distance big, sequence preservative, sequence two in species kind
There is preferable gene of the features such as conserved sequence can design universal primer as species identification and research spore relation at end.It is based on
The rapid identification method extensive uses in recent years of Mitochondria In Developing Flight Muscle of Insects COI genetic fragments, Lepidoptera, Hymenoptera, coleopteron
Quick detection kit also emerges in an endless stream, effectively the weak point supplemented with typoiogical classification.
The content of the invention
(1) technical problem to be solved
The technical problem to be solved in the present invention is how effectively to differentiate Douglas fir large small moth.
(2) technical scheme
In order to solve the above-mentioned technical problem or at least in part solve the above problems, be used to examine the invention provides one kind
The primer of Douglas fir large small moth is surveyed, the primer includes specific primer pair, and its nucleotides sequence is classified as：
Forward primer：5 '-AGCCCCAAGGATAGATGA-3 ', as shown in SEQ ID NO.1；
Reverse primer：5 '-AGGAATCCCAGGAAGACT-3 ', as shown in SEQ ID NO.2.
MtDNA COI genes of the invention to Douglas fir large small moth and other insect mtDNA COI genes announced on NCBI
Sequence is compared and screened, and the mtDNA COI gene inter-species homologys of Douglas fir large small moth is found up to more than 99%, with other
The mtDNA COI gene differences of insect are big, therefore the provable fragment has specificity to Douglas fir large small moth, can be very good area
Divide Douglas fir large small moth and other kind insects.
In a preferred embodiment of the invention, in order to be able to preferably expand COI genes and efficiently detect Douglas fir
Large small moth, also includes universal primer in the primer for detecting Douglas fir large small moth, and the nucleotide sequence of universal primer is preferably：
LCO1490：5 '-GGTCAACAAATCATAAAGATATTGG-3 ', as shown in SEQ ID NO.3；
HCO2198：5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 ', as shown in SEQ ID NO.4.
Present invention also offers a kind of kit containing above-mentioned primer.
Present invention also offers a kind of detection reagent containing above-mentioned primer.
Further, Taq enzyme, PCR Buffer and dNTP Mixture are also included in kit or detection reagent.
The kit or detection reagent include the primer for being used to detect Douglas fir large small moth, by primer, can efficiently examine
Survey Douglas fir large small moth.
Present invention also offers a kind of method for detecting Douglas fir large small moth, including：
(1) sample DNA is extracted；
(2) DNA using step (1) extraction enters performing PCR amplification anti-as template using above-mentioned forward primer and reverse primer
Should, wherein,
Forward primer：5 '-AGCCCCAAGGATAGATGA-3 ', as shown in SEQ ID NO.1；
Reverse primer：5 '-AGGAATCCCAGGAAGACT-3 ', as shown in SEQ ID NO.2；
(3) PCR primer is analyzed.
The detection method is labelled by nested-PCR method.
Further, in order to be able to preferably expand COI genes, step (2) is specially：
(21) DNA using step (1) extraction utilizes universal primer to carry out the amplification of first round PCR as template, expands purpose bar
Band is 650-750bp, preferably 700bp；
(22) 1 is pressed with first round pcr amplification product:Product after 50~100 dilutions is template, using forward primer and instead
Second, which is carried out, to primer takes turns PCR amplifications；
Wherein, universal primer is LCO1490 and HCO2198；The nucleotide sequence of forward primer and reverse primer is respectively such as
Shown in SEQ ID NO.1 and SEQ ID NO.2.
In a preferred embodiment of the invention, in step (21), PCR reaction systems are：It is calculated as with 25 μ L：0.125μ
L Taq enzymes, 2.5 μ 10 × PCR of L Buffer, 2 μ L dNTP Mixture, 1 μ L DNA, 1 μ L primers LCO1490,1 μ L primers
HCO2198 and 17.375 μ L sterile deionized waters.
Further, in the step (21), PCR reaction conditions are 94 DEG C of pre-degeneration 5min；94 DEG C are denatured 30s, 56 DEG C
Renaturation 30s, 72 DEG C of extension 1.5min, totally 35 circulations；72 DEG C of extension 5min.
In a preferred embodiment of the invention, in step (22), PCR reaction systems are：It is calculated as with 25 μ L：0.125μ
L Taq enzymes, 2.5 μ 10 × PCR of L Buffer, 2 μ L dNTP Mixture, 1 μ L DNA, 1 μ L forward primers, 1 μ L reverse primers
With 17.375 μ L sterile deionized waters.
Further, in the step (22), PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94 DEG C are denatured 30s, 50 DEG C
Renaturation 30s, 72 DEG C of extension 1.5min, 35 circulations；72 DEG C of extension 5min.
Wherein, in step (22), amplification ratio is preferably 1:50.
In a preferred embodiment of the invention, step (3) is：When PCR primer expand stripe size be 343bp,
Then result is the positive.
Present invention also offers application of the method for above-mentioned detection Douglas fir large small moth in plant protection.
The present invention can efficiently solve worm state during Douglas fir large small moth is intercepted and captured at port scene with Protocols in Molecular Biology
Differ the problem of being difficult to differentiate, and mitochondrial DNA COI sequences Designs according to measured by experiment and screened a pair of Douglas firs
The specific primer of large small moth, further checking has been carried out by real-time fluorescence PCR experiment to its specificity and sensitivity, real
Testing the Douglas fir large small moth specific primer of middle design specifically can distinguish Douglas fir large small moth with other bark beetles, wherein Douglas fir
The detection of large small moth is limited to 20pg, and other five kinds of bark beetles can not still be expanded in 20ng, illustrates the primer pair Douglas fir large small moth
Sensitivity it is at least higher than other species bark beetles 1000 times, specificity is good.
Brief description of the drawings
Fig. 1 is detection figure of the agarose gel electrophoresis in the embodiment of the present invention to five kinds of bark beetle first round pcr amplification products；
Wherein, swimming lane：1:U.S. pine tooth bark beetle；2:Larch bark bcetle；3:Northern knurl bark beetle；4:Douglas fir large small moth；5:Loose 12 teeth
Bark beetle；CK:Negative control；M:DL2000 Marker(100,250,500,750,1000,2000bp)；
Fig. 2 is detection figure of the agarose gel electrophoresis of the present invention to five kinds of bark beetles, 50 DEG C of second wheel pcr amplification product；Its
In, swimming lane：1:U.S. pine tooth bark beetle；2:Larch bark bcetle；3:Northern knurl bark beetle；4:Douglas fir large small moth；5:Loose 12 teeth are small
It is moth-eaten；CK:Negative control；M:DL2000 Marker(100,250,500,750,1000,2000bp)；
Fig. 3 is the amplification curve diagram of Douglas fir large small moth real-time quantitative PCR in the embodiment of the present invention；Wherein, marked in figure
20ng, 2ng, 200pg, 20pg are the addition of Douglas fir large small moth template in real-time quantitative PCR；
Fig. 4 is the template concentrations and C of real-time quantitative PCR reaction in the embodiment of the present inventionTBetween canonical plotting；
Fig. 5 is the melting curve figure of Douglas fir large small moth real-time quantitative PCR in the embodiment of the present invention.
Embodiment
With reference to embodiment, the embodiment to the present invention is described in further detail.Following examples are used for
Illustrate the present invention, but be not limited to the scope of the present invention.
Unless otherwise specified, the routine techniques hand that technological means used in embodiment is well known to those skilled in the art
Section.Unless otherwise specified, reagent used in embodiment is commercially available.
The nested PCR detection method of the Douglas fir large small moth of embodiment 1
(1) agents useful for same：EZgene Insect gDNA Kit kits：Purchased from BIOMIGA companies；SYBR Premix
Ex TaqⅡ(Perfect Real time)：Purchased from precious bioengineering (Dalian) Co., Ltd；DL2000 DNA Marker：Purchase
From precious bioengineering (Dalian) Co., Ltd；1×Loading Buffer：Purchased from precious bioengineering (Dalian) Co., Ltd.
(2) DNA is extracted：Targeted insect is extracted using the EZgene Insect gDNA Kit kits of BIOMIGA companies
The gDNA of (including U.S. loose tooth bark beetle, Larch bark bcetle, northern knurl bark beetle, Douglas fir large small moth and ips sexdentatus),
DNA is diluted to 50ng/ μ L.
(3) using gDNA as template, first round PCR amplification COI sequences, clip size are carried out with universal primer (table 1-1)
700bp。
Universal primer LCO1490 and the HCO2198 sequence of table 1
PCR reaction systems are as shown in table 2.
The PCR reaction systems of table 2
PCR reaction conditions are 94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 56 DEG C of renaturation 30s, 72 DEG C of extension 1.5min, altogether
35 circulations；72 DEG C of extension 5min.
(4) according to first round PCR testing result, choose positive PCR primer and be sequenced, examining order is by giving birth to the biological work of work
Journey (Shanghai) limited company completes.Sequencing result is handled, analyzed using bio-edit softwares, retains shared
Sequence carries out homologous comparison, and the obvious sequence of difference between Douglas fir large small moth and its sibling species is found out in contrast.By the Huang after processing
China fir large small moth sequence imports the specific primer that Douglas fir large small moth is designed in the softwares of Primer Premier 5.0, such as table 3.
(5) 50 times are diluted as template using first round PCR primer, carry out second with specific primer (table 3) and take turns PCR expansions
Increase, if clip size is 343bp, result is the positive.
The specific primer of the Douglas fir large small moth of table 3 is to sequence
PCR reaction systems are consistent with first round PCR reaction system, i.e., in terms of 25 μ L, 0.125 μ L TaKaRa Taq (5U/ μ
L), 2.5 μ 10 × PCR of L Buffer, 2 μ L dNTP Mixture (each 2.5mM), 1 μ L template DNAs, 1 μ L forward primers, 1 μ L
Reverse primer and 17.375 μ L sterile deionized waters.
PCR reaction conditions are 94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 50 DEG C of renaturation 30s, 72 DEG C of extension 1.5min, altogether
35 circulations；72 DEG C of extension 5min.
(6) with 5 kinds of insect genes groups DNA (1:U.S. pine tooth bark beetle；2:Larch bark bcetle；3:Northern knurl bark beetle；4:It is yellow
China fir large small moth；5:Ips sexdentatus) it is template, while setting negative control, examined according to the nested PCR method of above-mentioned foundation
Survey, as seen from Figure 1, expanded through first round PCR, the universal primer that this experiment is selected successfully can expand five kinds of bark beetles, product
Size about 700bp, illustrates the integrality of target area DNA sequence dna, is provided reliably for follow-up sequence alignment and appraisal
Ensure.
Fig. 2 is at 50 DEG C, second takes turns PCR amplifications, it can be clearly seen that is designed at 50 DEG C draws in Fig. 2
Thing can distinguish Douglas fir large small moth and other 4 kinds of bark beetles and control, meet the requirement of Rapid identification.
(7) Evaluation on specificity
The present invention verifies the specificity of the nest-type PRC primer of the present invention using real time fluorescence quantifying PCR method.
From the Real Time System of ABI PRISM 7500 reaction result, as shown in figure 3, Douglas fir large small moth
C under 20pgTValue is still less than 20, and C of other species bark beetles under 20ngTValue is all higher than 25, shows that Douglas fir large small moth exists
It can be amplified during 20pg, and other five kinds of bark beetles can not be amplified under 20ng, the specificity of primer is preferably.Such as Fig. 4 institutes
Show, standard curve result indicating template concentration and CTBe presented good correlation between value, the slope of standard curve of reaction for-
3.627, R2 be that 0.99, Eff% is 88.66, illustrates that the amplification efficiency of this experiment is higher, experimental result is reliable.Meanwhile, such as Fig. 5
Shown, melting curve is the quality control approach of whole course of reaction, and the melting curve of reaction is unimodal, without secondary peak and non-
Normal amplification, illustrates primer dimer negligible amounts, and PCR specificity is higher.
Finally, method of the invention is only preferably embodiment, is not intended to limit the scope of the present invention.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Sequence table
<110>Beijing Forestry University
<120>Detect primer, kit and the detection method of Douglas fir large small moth
<130> KHP171111002.4
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence
<400> 1
agccccaaggatagatga 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
aggaatcccaggaagact 18
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<400> 3
ggtcaacaaatcataaagatattgg 25
<210> 4
<211> 26
<212> DNA
<213>Artificial sequence
<400> 4
taaacttcagggtgaccaaaaaatca 26
Claims (10)
1. a kind of primer for being used to detect Douglas fir large small moth, it is characterised in that including：
Forward primer：5’-AGCCCCAAGGATAGATGA-3’；
Reverse primer：5’-AGGAATCCCAGGAAGACT-3’.
2. primer according to claim 1, it is characterised in that also including a pair of universal primers：
LCO1490：5’-GGTCAACAAATCATAAAGATATTGG-3；
HCO2198：5’-TAAACTTCAGGGTGACCAAAAAATCA-3.
3. the kit containing primer described in claim 1 or 2.
4. kit according to claim 3, it is characterised in that also include Taq enzyme, PCR Buffer in the kit
With dNTP Mixture.
5. a kind of method for detecting Douglas fir large small moth, it is characterised in that including：
(1) sample DNA is extracted；
(2) DNA using step (1) extraction enters performing PCR as template using the forward primer and reverse primer described in claim 1
Amplified reaction；
(3) PCR primer is analyzed.
6. method according to claim 5, it is characterised in that the step (2) is specially：
(21) DNA using step (1) extraction utilizes the universal primer described in claim 2 to carry out the expansion of first round PCR as template
Increase, amplification purpose band is 650-750bp；
(22) 1 is pressed with first round pcr amplification product:Product after 50~100 dilutions is template, using described in claim 1
Forward primer and reverse primer carry out second and take turns PCR amplifications.
7. method according to claim 6, it is characterised in that in step (21), PCR reaction systems are：In terms of 25 μ L,
0.125 μ L Taq enzymes, 2.5 μ 10 × PCR of L Buffer, 2.5 μ L dNTP Mixture, 1 μ L DNA, 1 μ L primers LCO1490,
1 μ L primers HCO2198 and 17.375 μ L sterile deionized waters.
8. the method according to claim 6 or 7, it is characterised in that in step (22), PCR reaction systems are：With 25 μ L
Meter, 0.125 μ L Taq enzymes, 2.5 μ 10 × PCR of L Buffer, 2 μ L dNTP Mixture, 1 μ L DNA, 1 μ L forward primers, 1 μ
L reverse primers and 17.375 μ L sterile deionized waters.
9. the method according to any one of claim 6-8, it is characterised in that PCR reaction conditions are in step (22)：94
DEG C pre-degeneration 5min；94 DEG C of denaturation 30s, 50 DEG C of renaturation 30s, 72 DEG C of extension 1.5min, 35 circulations；72 DEG C of extension 5min.
10. application of the method described in claim any one of 5-9 in plant protection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710448505.8A CN107164500B (en) | 2017-06-14 | 2017-06-14 | Primer, kit and detection method for detecting ips typographus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710448505.8A CN107164500B (en) | 2017-06-14 | 2017-06-14 | Primer, kit and detection method for detecting ips typographus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107164500A true CN107164500A (en) | 2017-09-15 |
| CN107164500B CN107164500B (en) | 2020-06-26 |
Family
ID=59818520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710448505.8A Expired - Fee Related CN107164500B (en) | 2017-06-14 | 2017-06-14 | Primer, kit and detection method for detecting ips typographus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107164500B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804664A (en) * | 2019-11-26 | 2020-02-18 | 拱北海关技术中心 | Primer pair and kit for identifying bark beetles in Meidiao and application of primer pair and kit |
| CN111500745A (en) * | 2020-04-29 | 2020-08-07 | 北京林业大学 | Long fragment amplification primer for eliminating interference of mitochondrion pseudogene of dendroctonus valens |
| CN111748638A (en) * | 2020-07-28 | 2020-10-09 | 南京海关动植物与食品检测中心 | Specific primer, kit and method for identifying ips insects based on DNA bar code |
| CN112921106A (en) * | 2021-04-22 | 2021-06-08 | 海口海关热带植物隔离检疫中心 | Real-time fluorescent PCR detection method for bark beetle of yellow fir, and primer and probe for detection |
| CN112941207A (en) * | 2021-04-22 | 2021-06-11 | 海口海关热带植物隔离检疫中心 | Real-time fluorescent PCR detection method for bark beetle of Pinus massoniana, and primer and probe for detection |
| CN113025724A (en) * | 2021-03-10 | 2021-06-25 | 华南农业大学 | Dual PCR primer, method and kit for identifying small pissodes punctatus |
| CN113025727A (en) * | 2021-04-22 | 2021-06-25 | 海口海关热带植物隔离检疫中心 | Real-time fluorescent PCR detection method for ips typographus and primer and probe for detection |
| CN113604586A (en) * | 2021-09-03 | 2021-11-05 | 上海市园林科学规划研究院 | Specific primer of bark beetle of black branch |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146811A (en) * | 2012-12-28 | 2013-06-12 | 北京林业大学 | Method to detect dendroctonus valens and detection kit |
| CN104232634A (en) * | 2014-09-25 | 2014-12-24 | 北京林业大学 | SS-COI (Sirex noctilio F. specific cytochrome oxidase I) primer pair and quick molecular detection method |
-
2017
- 2017-06-14 CN CN201710448505.8A patent/CN107164500B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146811A (en) * | 2012-12-28 | 2013-06-12 | 北京林业大学 | Method to detect dendroctonus valens and detection kit |
| CN104232634A (en) * | 2014-09-25 | 2014-12-24 | 北京林业大学 | SS-COI (Sirex noctilio F. specific cytochrome oxidase I) primer pair and quick molecular detection method |
Non-Patent Citations (2)
| Title |
|---|
| ENRICO A. RUIZ等: "Molecular and Morphological Analysis of Dendroctonus pseudotsugae (Coleoptera: Curculionidae: Scolytinae): An Assessment of the Taxonomic Status of Subspecies", 《ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA》 * |
| 殷玉生等: "基于线粒体COI基因鉴定大小蠹属昆虫", 《福建林业科技》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804664A (en) * | 2019-11-26 | 2020-02-18 | 拱北海关技术中心 | Primer pair and kit for identifying bark beetles in Meidiao and application of primer pair and kit |
| CN111500745A (en) * | 2020-04-29 | 2020-08-07 | 北京林业大学 | Long fragment amplification primer for eliminating interference of mitochondrion pseudogene of dendroctonus valens |
| CN111500745B (en) * | 2020-04-29 | 2022-04-26 | 北京林业大学 | Long fragment amplification primer for eliminating interference of mitochondrion pseudogene of dendroctonus valens |
| CN111748638A (en) * | 2020-07-28 | 2020-10-09 | 南京海关动植物与食品检测中心 | Specific primer, kit and method for identifying ips insects based on DNA bar code |
| CN113025724A (en) * | 2021-03-10 | 2021-06-25 | 华南农业大学 | Dual PCR primer, method and kit for identifying small pissodes punctatus |
| CN112921106A (en) * | 2021-04-22 | 2021-06-08 | 海口海关热带植物隔离检疫中心 | Real-time fluorescent PCR detection method for bark beetle of yellow fir, and primer and probe for detection |
| CN112941207A (en) * | 2021-04-22 | 2021-06-11 | 海口海关热带植物隔离检疫中心 | Real-time fluorescent PCR detection method for bark beetle of Pinus massoniana, and primer and probe for detection |
| CN113025727A (en) * | 2021-04-22 | 2021-06-25 | 海口海关热带植物隔离检疫中心 | Real-time fluorescent PCR detection method for ips typographus and primer and probe for detection |
| CN113604586A (en) * | 2021-09-03 | 2021-11-05 | 上海市园林科学规划研究院 | Specific primer of bark beetle of black branch |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107164500B (en) | 2020-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107164500A (en) | Detect primer, kit and the detection method of Douglas fir large small moth | |
| Keremane et al. | A rapid field detection system for citrus huanglongbing associated ‘Candidatus Liberibacter asiaticus’ from the psyllid vector, Diaphorina citri Kuwayama and its implications in disease management | |
| Böhm et al. | Real‐time quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants | |
| Duan et al. | Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification | |
| Osman et al. | Real-time RT-PCR (TaqMan®) assays for the detection of viruses associated with Rugose wood complex of grapevine | |
| Wozney et al. | Real-time PCR detection and quantification of elephantid DNA: Species identification for highly processed samples associated with the ivory trade | |
| Gonthier et al. | A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers | |
| CN105331743A (en) | Kit for rapidly detecting various viruses in muskmelon by one step and rapid detection method adopting kit | |
| Marbot et al. | Development of real-time RT-PCR assay for detection of Prunus necrotic ringspot virus in fruit trees | |
| Guerrero et al. | Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas | |
| Leal et al. | Development of two reverse transcription‐PCR methods to detect living pinewood nematode, B ursaphelenchus xylophilus, in wood | |
| Przybylska et al. | PCR-RFLP method to distinguish Frankliniella occidentalis, Frankliniella intonsa, Frankliniella pallida and Frankliniella tenuicornis | |
| CN101864476B (en) | Method for identifying coilia ectenes and coilia mystus by using PCR | |
| Bracalini et al. | Alien pest molecular diagnostics: can DNA traces be exploited to assess the damage caused by the western conifer seed bug on stone pine fructification? | |
| CN102559922B (en) | Molecular biological method for distinguishing striped rice borer from chilotraea auricilia dudgeon | |
| CN110592269A (en) | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) | |
| KR102173154B1 (en) | Loop Mediated Isothermal Amplification Primer Set for Detection of Dengue Virus Serotype 1 or 3 and Uses Thereof | |
| CN109355396B (en) | PCR detection specific primer and kit for Meloidogyne haplocalyx | |
| CN109486960B (en) | Method for detecting Meloidogyne incognita by applying RPA technology, RPA primer and kit | |
| CN107663547B (en) | PCR (polymerase chain reaction) rapid detection method for frankliniella occidentalis based on specific SS-COI (sequence specific-polymerase chain reaction) primer | |
| CN110894551A (en) | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type I virus (GCRV-I) | |
| Kumar et al. | Detection of specific nucleotide changes in the hypervariable region of 16S rDNA gene of Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum | |
| CN113215288B (en) | Pine wood nematode disease nested PCR detection kit and use method thereof | |
| CN111518938B (en) | CAPs molecular marker for identifying single seed of Amaranthus praecox, Amaranthus praecox and Amaranthus sanderi based on SNP locus and application thereof | |
| KR100639834B1 (en) | Molecular marker for the detection of A1 mating type of Phytophthora infestans |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200626 |