CN108866240A - For identifying primer and enzyme and its application of DAdV-3 and DAdV-A - Google Patents
For identifying primer and enzyme and its application of DAdV-3 and DAdV-A Download PDFInfo
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Abstract
The present invention is provided to identify the primer of DAdV-3 and DAdV-A and enzyme and its application, belong to duck disease research field, use I enzyme of the primer as shown in SEQ ID NO.1-2 and Sal, establishing can be to the two kinds of Ana 1 aviadenovirus occurred in China duck group（DAdV-3 and DAdV-A）Differential diagnostic method, the sensibility and PCR of this method are suitable, it only needs to carry out conventional agarose gel electrophoresis again after carrying out additional 10min digestion to PCR product, it is simple and practical, convenient and efficient, support is provided to carry out adenovirus Molecular Epidemic poison investigation epidemic disease related to science bridle in duck group, there is highly important research significance.
Description
Technical field
The invention belongs to duck disease research fields, and in particular to for identify DAdV-3 and DAdV-A primer and enzyme and its
Using.
Background technique
Adenovirus particles are no cyst membrane, nucleocapsid is in icosahedral symmetry, linear, double-stranded DNA virus, full-length genome
About 33kd or so.Wherein, hexon（Hexon）It is that Adenoviridae respectively belongs to the most important structural proteins of virus, each
Hexon protein surface carries the species-specific antigen determinant of the main category of virus, is the target of virucidin, contains
There is the main protective antigen gene cluster of virus, it is pathogenic closely related with virus.
According to International Commission on Virus Classification（International Committee on Taxonomy of
Viruses, ICTV）Newest classification（https://talk.ictvonline.org/）, Adenoviridae（Family:Adenoviridae）Divide into 5 categories（Genera）, respectively richness AT Adenovirus (Genus:Atadenovirus), fowl adenopathy
Poison belongs to (Genus:Aviadenovirus), fish Adenovirus (Genus:Ichtadenovirus), mastadenovirus
(Genus:) and sialidase Tobamovirus (Genus Mastadenovirus:Siadenovirus).
The study found that there are a variety of adenovirus infections in birds, wherein the adenovirus of infected chicken has 5 kinds：Fowl adenovirus A
（Fowl aviadenovirus A, FAdV-A）, aviadenovirus Type B（Fowl aviadenovirus B, FAdV-B）, fowl adenopathy
Malicious c-type（Fowl aviadenovirus C, FAdV-C）, aviadenovirus D type（Fowl aviadenovirus D, FAdV-D）,
Aviadenovirus E type（Fowl aviadenovirus E, FAdV-E）, belong to Aviadenovirus.Infect turkey（Turkey）'s
Adenovirus has 4 kinds, is belonging respectively to two categories of Adenoviridae, wherein turkey adenovirus Type B（Turkey aviadenovirus
B）, turkey adenovirus c-type（Turkey aviadenovirus C）With turkey adenovirus D type（Turkey aviadenovirus
D）Belong to Aviadenovirus；And turkey sialidase virus type A（Turkey siadenovirus A）Belong to sialidase virus
Belong to.The adenovirus of infection parrot (Psittacine) has 2 kinds, is belonging respectively to two categories of Adenoviridae, wherein parrot richness AT
Adenovirus A type（Psittacine atadenovirus A）Belong to rich AT Adenovirus；And parrot adenovirus Type B
（Psittacine aviadenovirus B）Belong to Aviadenovirus.
In recent years, with China aquatic bird（Especially duck）The continuous expansion of raising scale, the development of aquaculture model, in aquatic bird
New hair epidemic disease is also more and more, becomes increasingly complex.There is also adhere to Adenoviridae difference Adenovirus separately for adenovirus infection in duck group
Relevant report, have the Ana 1 aviadenovirus A type for belonging to rich AT Adenovirus（Duck adenovirus A, is abbreviated as DAdV-A）.
2014, with the development of high-throughput techniques, Marek A etc. determined 1 plant of duck source adenovirus（GR plants, GenBank accession number
NC024486）Whole genome sequence, and it is named as 2 type of Ana 1 aviadenovirus（Duck adenovirus 2, DAdV-2）, and base
Aviadenovirus member is classified as in genome characteristics and genetic evolution feature（Marek A, Kaján GL, Kosiol C,
et al. Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2
extend the number of species within the genus Aviadenovirus[J]. Virology,
2014, 462-463: 107-114.）, its scientific is Ana 1 aviadenovirus Type B by subsequent ICTV（duck aviadenovirus
B, to distinguish the DAdV-A for belonging to rich AT Adenovirus）.Then, since 2014, occurred a kind of new duck in China kind duck
Adenovirus（CH-GD-2014 plants, GenBank accession number KR135164）, there are following bright for GR plants of the virus and DAdV-2 separation strains
Aobvious difference：1. its whole genome sequence and GR plants of nucleotide homology are only 92.3%；2. CH-GD-2014 plants and GR plants
The nucleotide homology of Hexon gene is only 76.6%, is lower than 80.0%；3. CH-GD-2014 plants have 2 spike proteins（Fiber
1,Fiber 2）, and GR plants of only 1 spike protein（Fiber）.Shown based on features described above popular a kind of new in China duck group
Ana 1 aviadenovirus, and be named as 3 type of Ana 1 aviadenovirus（Duck adenovirus 3, DAdV-3）（Zhou Zhenhai Ana 1 aviadenovirus 3
Type genomic sequence analysis and Study on Pathogenicity [D] Agricultural University Of South China Master's thesis, 2016.）.This just give DAdV-3 and
DAdV-A brings current demand, it is however generally that, antidiastole is carried out to two cause of diseases, needs to be directed to separately designing for 2 cause of diseases
Primer（4 primers are needed altogether）, to establish differential diagnostic method.Hexon gene of this research based on DAdV-3 and DAdV-A
Conservative region nucleotide sequence feature, establish it is a kind of only need 2 primers can DAdV-3 and DAdV-A carry out while expanding, but expanding
Increasing region, there are I enzyme polymorphism differences of Sal（I.e. there are I enzyme restriction enzyme site differences of Sal in PCR amplification region）, to digestion products into
Row conventional agarose gel electrophoresis can be distinguished effectively.The sensibility and PCR of this method are suitable, only need to PCR product into
The additional 10min digestion of row（Without carrying out glue recycling to product）Carry out electrophoresis again afterwards, it is simple and practical, convenient and efficient, it especially can be right
DAdV-3 and DAdV-A mixed infection is precisely distinguished, currently there is not yet the mirror of DAdV-3 and DAdV-A based on similar principles
The report of the primer and enzyme that do not diagnose, this research can fill up Related Research Domain blank.
Summary of the invention
The purpose of the present invention is to provide the primer provided for identifying DAdV-3 and DAdV-A and enzyme and its applications, establish
One kind only need 2 primers can DAdV-3 and DAdV-A carry out while expanding, but there are I enzyme polymorphism of Sal is poor in amplification region
It is different（I.e. there are I enzyme restriction enzyme site differences of Sal in PCR amplification region）, conventional agarose gel electrophoresis is carried out to digestion products
Effectively distinguish.
Following technical scheme is used to achieve the above object：
One group for identifying the primer and enzyme of DAdV-3 and DAdV-A, the primer is as follows：
DAdV-3A-F1：5 '-CATACMGYGGMACAGCTTACAATC-3 ',
DAdV-3A-R1：5'-ATCSCKAAAACCWATGTAGTTA-3'；
The enzyme is I enzyme of Sal.
It is a further object of the present invention to provide a kind of for detecting the kit of DAdV-3 and DAdV-A, draws including described
Object and enzyme.
It is expanded according to the 50 μ L systems that DreamTaq Green PCR Master Mix (2 ×) kit is recommended,
Wherein 2 × PCR Master Mix amplification reaction solution, 25 μ L, 10 μM of primers DAdV-3A-F1 and DAdV-3A-R1 are respectively 1.0
μ L, extraction it is nucleic acid-templated（DAdV-3,DAdV-A）Each 1.0 μ L, supplement sterile deionized water to 50 μ L of final volume, after mixing
Carry out PCR amplification.
Amplification condition enters circulation, 95 DEG C of denaturation 30 s, △ T after being 95 DEG C of 3 min of initial denaturation（52 ℃-62 ℃）
Anneal 30 s, 72 DEG C of extension 35s, and 40 after circulation terminates, and 72 DEG C extend 10 min eventually, after reaction according to conventional agar
Sugared gel electrophoresis identification.△T（52 ℃-62 ℃）Indicate that annealing temperature optimizes in this section.Most preferably moving back after optimized
Fiery temperature is 56 DEG C.
The advantage of the invention is that：
The present invention is based on the Hexon genes of DAdV-3 and DAdV-A to guard region nucleotide sequence feature, and establishing one kind only needs 2 to draw
Object can DAdV-3 and DAdV-A carry out while expanding, but there are I enzyme polymorphism differences of Sal in amplification region（That is PCR amplification area
There are I enzyme restriction enzyme site differences of Sal in domain）, carrying out conventional agarose gel electrophoresis to digestion products can effectively distinguish.This method
Sensibility and PCR it is suitable, only need to carry out electrophoresis, simple and practical, side again after carrying out PCR product additional 10min digestion
Just quick, especially DAdV-3 and DAdV-A mixed infection can precisely be distinguished, currently there is not yet based on similar principles
The primer of the antidiastole of DAdV-3 and DAdV-A and the report of enzyme, this research can fill up Related Research Domain blank.
Detailed description of the invention
Fig. 1 upstream primer designs area.
Fig. 2 downstream primer designs area.
Fig. 3 459-465 nucleotide sequence comparisons of DAdV-3 and DAdV-A.
The electrophoresis of Fig. 4 PCR product, wherein M：DL2000 molecular weight standard；1：DAdV-3；2：DAdV-A.
Fig. 5 specific test；M：DL2000 molecular weight standard；1：DAdV-3；2：DAdV-A；3：DEV；4：MDPV；5：
DuCV；6：GPV；7：E. coli；8：R.A.；9：P.M.；10：DAdV-2；11：Sterile deionized water.
Product electrophoresis after Fig. 6 Sal I enzyme digestion identification, wherein M1：DL500 molecular weight standard；M2：DL2000 molecule
Amount standard；1：DAdV-3 product restriction enzyme digestion and electrophoresis；2：DAdV-A product restriction enzyme digestion and electrophoresis；3：DAdV-3 and DAdV-A coinfection product enzyme
Cut electrophoresis.
Specific embodiment
Embodiment 1
1, material
1.1 strains and bacterial strain
Test cause of disease DAdV-3（W1FJ plants, GenBank accession number MH349774）,DAdV-A（FJ12025 plants, GenBank is stepped on
Record KF286430）It is saved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.
Test control strain duck enteritis virus（DEV）, Muscovy duck parvovirus（MDPV）, duck circovirus（DuCV）, duck
Source goose parvovirus（GPV）；Test control strain E. coli isolated from ducks（E. coli）, Riemerlla anatipestifer (R.A.) He Yayuan
Eggs crack detection (P.M.), is saved by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute.2 type of Ana 1 aviadenovirus
（DAdV-2）Hexon gene（GR plants, GenBank accession number NC024486）Sequence is by giving birth to work bioengineering (Shanghai) stock
The synthesis of part Co., Ltd.
1.2 design of primers
According to National Center for Biotechnology Information (National Center of Biotechnology
Information, NCBI）Adenoviridae DAdV-3 representative strains on database GenBank：CH-GD-2014 plants（KR135164）,
W1GD plants（MH349773）, W1FJ plants（MH349774）, W1AH plants（MH349775）With DAdV-A representative strains：E05 plants
（EF532411）, FJ12025 plants（KF286430）, D11-JW-012 plants（KJ452170）, D11-JW-017 plants（KJ452171）
Hexon gene, using Lasergene DNAStar MegAlign carry out nucleotide series analyses and comparison, determine DAdV-3 and
DAdV-A gene conserved region is respectively as upstream primer design area (Fig. 1) and downstream primer design area (Fig. 2).
A set of primer is designed using primer-design software Oligo 7.0, it is as follows：
DAdV-3A-F1：5 '-CATACMGYGGMACAGCTTACAATC-3 ',
DAdV-3A -R1：5'-ATCSCKAAAACCWATGTAGTTA-3'.Above-mentioned primer is by raw work bioengineering (Shanghai) stock
The synthesis of part Co., Ltd.
It should be pointed out that when being expanded using DAdV-3A-F1/ DAdV-3A-R1 to DAdV-3 and DAdV-A,
The PCR product purpose band size of DAdV-3 and DAdV-A is respectively 524bp and 521bp（Conventional agarose electrophoresis, visually can not
Effectively distinguish）.But there are characteristic I enzyme restriction enzyme site differences of Sal in the amplification region of DAdV-3 and DAdV-A, wherein
DAdV-3 is classified as GTCGAC in 459-465 nucleotides sequences（It can be identified by I enzyme of Sal）, and DAdV-A does not have in amplification region
There is the position that can be identified by I enzyme of Sal, sequence alignment result is shown in Fig. 3.
1.3 main agents
DreamTaq Green PCR Master Mix (2 ×) is purchased from ThermoScientific company, EasyPure
Genomic DNA Kit, that EasyPure Bacteria Genomic DNA Kit is purchased from the full formula gold biotechnology in Beijing is limited
Company；DNA molecular amount standard DL500, I enzyme of DNA molecular amount standard DL2000 and QuickCut Sal are purchased from Bao doctor's object
Technology (Beijing) Co., Ltd.
The foundation of test method
The extraction of 2.1 genomic DNAs
DAdV-3, DAdV-A, DEV, MDPV, DuCV, GPV are mentioned according to the method for EasyPure Genomic DNA Kit kit
Take corresponding genomic DNA, -80 DEG C freeze it is spare.
E. coli, R.A. and P.M. according to EasyPure Bacteria Genomic DNA Kit kit method
Extract corresponding genomic DNA, -80 DEG C freeze it is spare.
The configuration of 2.2 reaction solutions and the optimization of annealing temperature
It is expanded according to the 50 μ L systems that DreamTaq Green PCR Master Mix (2 ×) kit is recommended, wherein
2 × PCR Master Mix amplification reaction solution, 25 μ L, primer DAdV-3A-F1 and DAdV-3A-R1（10μM）Respectively 1.0 μ
L, extraction is nucleic acid-templated（DAdV-3,DAdV-A）Each 1.0 μ L, supplement sterile deionized water to 50 μ L of final volume, mix laggard
Row PCR amplification.
Amplification condition enters circulation, 95 DEG C of denaturation 30 s, △ T after being 95 DEG C of 3 min of initial denaturation（52 ℃-62 ℃）
Anneal 30 s, 72 DEG C of extension 35s, and 40 after circulation terminates, and 72 DEG C extend 10 min eventually, after reaction according to conventional agar
Sugared gel electrophoresis identification.△T（52 ℃-62 ℃）Indicate annealing temperature optimized in this section, it is optimized after most preferably move back
Fiery temperature is 56 DEG C.
As a result visible（Fig. 4）, when only DAdV-3 is added in template, there is the band that size is 524bp（1st swimming lane）；When
When DAdV-A is only added in template, there is the band that size is 521bp（2nd swimming lane）, conventional agarose electrophoresis naked eyes cannot be distinguished.
2.3 specific test
By the PCR system and amplification condition after optimization, to DEV, MDPV, DuCV, GPV, E. coli, R.A. and P.M. and go out
The control of bacterium deionized water is expanded, and amplified band is showed no.As a result see Fig. 5, DEV（3rd swimming lane）,MDPV（4th swimming lane）,
DuCV（5th swimming lane）,GPV（6th swimming lane）,E. coli（7th swimming lane）,R.A.（8th swimming lane）And P.M.（9th swimming lane）,
DAdV-2（10th swimming lane）And sterile deionized water（11st swimming lane）, show the method high specificity established, to common aquatic bird disease
Former equal no cross reaction.
2.4 digestions identification
PCR product is subjected to digestion identification using I enzyme of Sal, digestion system is 30 μ L systems：Wherein 10 × QuickCut Green
3 μ L of Buffer, 3 μ L of QuickCut Sal I enzyme, 20 μ L of PCR product, supplement sterile deionized water to 30 μ L of final volume.Gently
Brief centrifugation after light mixing, 37 DEG C of 10 min of water-bath.As a result visible Fig. 6, DAdV-3 product have two, and size is respectively
101bp and 423bp（1st swimming lane）；DAdV-A primer size is constant, is still 521bp（2nd swimming lane）；DAdV-3 and DAdV-A
Product has three, and size is respectively 521bp, 423bp and 101bp（3rd swimming lane）.
Clinical application
After 49 parts of clinical inspection duck source pathological material of diseases conventionally milled processed, EasyPure Genomic DNA Kit is utilized
Corresponding nucleic acid DNA is extracted, the detection of DAdV-3 and DAdV-A infection is carried out using the method after optimization.PCR product is through Sal I
Conventional agarose gel electrophoresis is carried out after enzyme digestion, as a result as it can be seen that detecting that 9 parts of DAdV-3 infection are positive, positive rate is
18.37%；Detect that 5 parts of DAdV-A infection are positive, positive rate 10.20%；Detect 2 parts of DAdV-3 and DAdV-A coinfection sun
Property, positive rate 4.08%.
The positive target fragment of PCR after reaction is utilized into Ago-Gel QIAquick Gel Extraction Kit（DP209, Tiangeng are biochemical
Science and technology（Beijing）Co., Ltd）Gel extraction is carried out respectively.Connection examination is cloned according to pEASY-T1 Simple Cloning Kit
Agent box（CT111-01, Beijing Quanshijin Biotechnology Co., Ltd）Specification willHexonGene fragment clone is to pEASY-T1
On carrier, 8 single bacteriums of random picking fall within ampicillin（Content is 100 μ g/mL）The LB liquid medium culture 14 of resistance
After h, the small extraction reagent kit of rapid plasmid is utilized（DP105, Tiangeng biochemical technology（Beijing）Co., Ltd）Extract corresponding plasmid.It adopts
Primer and condition when with PCR amplification carry out PCR identification to the plasmid of extraction respectively, send the positive recombinant plasmid filtered out to treasured
Day cures biotechnology（Beijing）Co., Ltd is sequenced.Sequencing result is carried out to BLAST analysis verifying on NCBI, is phase
The Hexon genetic fragment of the DAdV-3 and DAdV-A that answer, wherein DAdV-3 positive fragment and DAdV-3（W1FJ plants, GenBank is stepped on
Record MH349774）Hexon genetic homology 99.0% or more；DAdV-A positive fragment and DAdV-A（FJ12025 plants,
GenBank accession number KF286430）Hexon genetic homology 99.0% or more.Above-mentioned sequencing result and PCR testing result
Coincidence rate be 100%.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute
<120>For identifying primer and enzyme and its application of DAdV-3 and DAdV-A
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence
<400> 1
catacmgygg macagcttac aatc 24
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
atcsckaaaa ccwatgtagt ta 22
Claims (2)
1. one group for identifying the primer and enzyme of DAdV-3 and DAdV-A, it is characterised in that：The primer is as follows：
DAdV-3A-F1：5 '-CATACMGYGGMACAGCTTACAATC-3 ',
DAdV-3A-R1：5'-ATCSCKAAAACCWATGTAGTTA-3'；The enzyme is I enzyme of Sal.
2. a kind of for detecting the kit of DAdV-3 and DAdV-A, which is characterized in that the kit includes claim 1 institute
The primer and enzyme stated.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
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| CN107058634A (en) * | 2017-06-13 | 2017-08-18 | 福建省农业科学院畜牧兽医研究所 | The type of Ana 1 aviadenovirus 2 and Ana 1 aviadenovirus A type double PCR detection primers and kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108842004A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | Distinguish real-time fluorescence quantitative PCR primer and its differentiating method and the application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
| CN108842005A (en) * | 2018-07-28 | 2018-11-20 | 福建省农业科学院畜牧兽医研究所 | The MGB probe and its mating primer and method of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B are detected simultaneously |
| CN108950070A (en) * | 2018-07-28 | 2018-12-07 | 福建省农业科学院畜牧兽医研究所 | Identify the PCR primer and its discrimination method and application of Ana 1 aviadenovirus c-type and Ana 1 aviadenovirus Type B |
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