CN107475445A - Differentiate kit and its application of chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT PCR and RFLP technology - Google Patents
Differentiate kit and its application of chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT PCR and RFLP technology Download PDFInfo
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Abstract
The invention discloses a kind of kit and its application for differentiating chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT PCR and RFLP technology.Described kit contains the primer pair and SpeI, SacI and StuI restriction enzyme for expanding chicken infectivity bursa of Fabricius virus VP 2 gene.The differential diagnostic method based on RT PCR and RFLP technologies that the present invention is established can distinguish IBDV highly virulent strain and low virulent strain, and step is simply easy to operation, proposition of the invention provides a kind of new effective technology means for the rapid differential diagnosis of IBDV highly-wetting liquids.
Description
Technical field
The present invention relates to a kind of kit for differentiating chicken infectivity bursa of Fabricius virus highly-wetting liquid and its application, especially relate to
A kind of and kit and its application for differentiating chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT-PCR and RFLP technologies.This
Invention belongs to virus detection techniques field.
Background technology
Bursal Disease (Infectious bursal disease, IBD) is by infectious bursal disease virus
(Infectious bursal disease virus, IBDV) causes the acute of chicken, high degree in contact, kills lymphatic biography
Catch an illness.IBD main infections object is 3~6 week old chickens, main to encroach on the central immune organ bursa of farbricius, causes immunosupress.IBDV
There are two kinds of different types, referred to as serum I type and serum II type.Serum I type virus is caused a disease to chicken；Serum II type is from turkey
Separation, to chicken had no pathogenicity.Virulence and duplicating efficiency of the IBDV serum I type separation strains in bursa of farbricius cell have different journeys
Degree.It is viral to be had differences in proliferation rate and on pathogenic, it can be divided into classical strainses or attenuated vaccine strain (cIBDV) by virulence, become
The different type such as different strain (vIBDV) and highly virulent strain (vvIBDV).
Chick vaccine inoculation is to control IBD main prevention and control strategy.But variation strain and highly virulent strain go out in recent years
It is existing, this sick generation and prevalence the characteristics of new is occurred again, so as to aggravate the harm to aviculture.Domestic and foreign scholars pair
IBD preventing and treating has carried out substantial amounts of research work, develops and develops a variety of vaccines, but application is most common or traditional weak
Malicious vaccine, but highly virulent strain occur after just expose many problems.Due to failing effectively to control virulent to spread, this sick morbidity
Rate and the death rate significantly raise, caused by economic loss it is huge.Therefore establish a kind of quick, sensitivity, accurately distinguish strong poison
Will be very crucial with prevention and control of the weak malicious diagnostic techniques to Bursal Disease.
In the last few years, had been applied to for IBDV various diagnostic techniques in differentiation and diagnosis to IBDV strains.
ELISA experiments, virus neutralization tests (VNT), real-time RT-PCR and agar-gel precipitation test (AGPT) had been used to examine
Various IBDV strains are surveyed and distinguished, but lack sensitive, special and quick differentiation highly virulent strain and low virulent strain, especially area
Divide infection and immune clinical testing procedure.And RT-PCR combination restriction enzymes enzyme assay method inherently has simple and fast
And the advantages that accurate, it is a kind of IBDV detection methods being worthy to be popularized.This detection method, from sampling, RNA extractions, RT-PCR, enzyme
Cut, product gel electrophoretic analysis, it is whole only to need the extremely short time to complete.This method is not only able to detect the chicken artificially infected
Embryo allantoic liquid and cell culture, and clinical sample can be directly detected, so, this method is a kind of special, quick, quick
Sense and practical IBD detection techniques.Therefore, the diagnostic techniques for distinguishing IBDV highly-wetting liquids is established, to obtain IBDV epidemiology
Information provides basis, also controls the disease to provide strong help to formulate vaccination strategies.
The content of the invention
It is an object of the invention to provide a kind of kit for being capable of rapid differential diagnosis IBDV highly virulent strains and low virulent strain
And its detection method.
In order to achieve the above object, present invention employs following technological means：
The present invention have chosen from GenBank 8 plants of IBDV highly virulent strains D6948, Gx of representative morbidity, IM, KKI,
KSH, OKYM, SH95 and UK661,6 plants of classical low virulent strain ZJ2000, B87, CEF94, D78, HZ-2 and JD-1 carry out sequence point
Analysis.The nucleotide sequence of above-mentioned 14 plants of strains is compared by DNAMAN softwares, determines the difference of IBDV highly-wetting liquid sequences
It is different to be concentrated mainly in the important Viral structural protein VP2 gene 860bp~1185bp regions of IBDV, pass through NEBcutter softwares
The specific cleavage site at base mutation position in highly-wetting liquid sequence difference concentrated area is analyzed, finally determines SpeI, SacI
With StuI as difference IBDV highly virulent strains and the restriction enzyme of low virulent strain.The purpose fragment of highly virulent strain can be by
SpeI cuts out 531bp and 302bp two sections of fragments, and 242bp and 591bp two sections of fragments can also be cut out by StuI, and can not
By SacI digestions；And the purpose fragment of low virulent strain only carries out digestion by SacI, 218bp and 615bp two sections of fragments are cut out.Cause
This, the present invention is by according to the Restrictive fragment length polymorphism of this section of VP2 target gene (restriction fragment
Length polymorphism, RFLP) sequence the characteristics of, with this section of VP2 genes combination restriction enzyme SpeI, SacI and
StuI establishes the method based on RT-PCR and RFLP technology for detection IBDV highly-wetting liquids.
General purpose I BDV identification primer L2, the U2 recommended first using OIE, using RT-PCR method, expands and identifies this reality
CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8,05-6,99-3, JC-7, SD-F3 and the GC-7 for testing room preservation are total to
15 plants of IBDV strains, as a result display can expand the purpose fragment to about 604bp, and sequencing result shows to be IBDV strains.Again
VP2 genes common conservative region design pair of primers PAU, PAD according to IBDV highly-wetting liquids, IBDV is expanded by RT-PCR
The common 833bp of VP2 gene 521bp~1354bp purpose fragment, this section of gene include highly-wetting liquid base mutation and mainly collected
The gene order in middle region；Further to identify the strong and weak characteristic of each strain virulence, using tri- kinds of limitations of SpeI, SacI and StuI
Property restriction endonuclease digestion identification is carried out to PCR primer after purification, wherein HR, CF, SD, DB11, BC and DN-04 can cut out by SpeI
531bp and 302bp two sections of fragments, can also be cut out 242bp and 591bp two sections of fragments by StuI, and can not by SacI digestions,
It is IBDV highly virulent strains to confirm this 6 plants；CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 are only by SacI
Cut out 218bp and 615bp two sections of fragments, it was demonstrated that this 9 plants are IBDV low virulent strains.
To verify the pathogenic of the IBDV highly-wetting liquids of the method identification of this research foundation, 15 strain virus were through 10 days by more than
After the chicken embryo rejuvenation of age SPF, the degree of purity of virus is detected using blood coagulation tests；Pass through the real-time fluorescence quantitative RT-PCR side established
Method determines the copy number of each strain virus, and the virus after quantifying is diluted with sterilizing PBS, and 15 strain virus are adjusted to
108.747/ mL, carry out eye droppings with 40 age in days SPF chickens and attack poison, every 100uL, control group is with PBS eye droppings, every group of 6 SPF chickens.Attack
48h after poison, each group chicken of infection HR, CF, SD, DB11, BC and DN-04 strain virus show the depressed, feed intake of spirit reduce,
Feather is fluffy and disorderly, head and wing are sagging, a dispirited, slow-witted vertical a corner and catacleisis, and the clinical symptoms such as be reluctant to walk about；PBS control
Group chicken is acted normally, and the chicken of other weak poison groups is without obvious clinical symptoms.The 3rd day after infection, every group randomly selects 3 chickens and carries out
Cut open and kill, different degrees of characteristic lesion occurs for each group chicken bursa through cut open inspection 15 strain virus of visible infection, wherein strong poison
Group chicken observes the obvious of the weaker poison group of lesion, as the bursa of farbricius is presented yellow gel-shaped oedema, covered with cream color on mucous membrane
Fibrinous exudate；Spleen enlargement, there is individually bleeding；There is striated and gone out in the Flaccid Coelogyne inner side muscle of the strong poison of infection
Blood.Lesion is not found after PBS control group cut open inspection.The bursa of farbricius and spleen are taken, carries out the making of paraffin section, PBS control group Fa Shi
Capsule and spleen tissue structure tend to be normal, do not observe obvious Histopathologic changes；6 plants of highly virulent strain HR, CF, SD, DB11,
BC and DN-04 strains bursa of farbricius pathologic examination after poison is attacked, it is seen that substantial amounts of lymphocyte necrosis and with apoptosis phenomenon,
There is substantial amounts of different preferendum cellular infiltration in lymph follicle, visible vacuolization in the folliculus of part, lamina propria oedema, have in lamina propria
Inflammatory cell infiltration.9 plants of low virulent strain CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strain methods after poison is attacked
Family name's capsule pathologic examination, it is seen that lymph follicle atrophy, lymphocyte therein substantially reduce, and have in lamina propria inflammatory thin
Born of the same parents infiltrate.This 15 plants of strain spleen pathologic examinations after poison is attacked, have no significant difference, between highly virulent strain and low virulent strain
It can be seen that lymphocyte significantly reduces, and white space is formed with substantial amounts of macrophage hyperplasia.Therefore, returned by animal real
The RT-PCR and RFLP method established in the real present invention of checking can quickly distinguish HR, CF of this laboratory preservation, SD,
DB11, BC and DN-04 strain are IBDV highly virulent strains, and CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strain are
IBDV low virulent strains, the discriminating available for IBDV highly virulent strains and low virulent strain detect.
On the basis of the studies above, the present invention proposes a kind of based on the avian infectious method of RT-PCR and RFLP technologies discriminating
The kit of family name's bursal disease virus highly-wetting liquid, described kit contain for expanding chicken infectivity bursa of Fabricius virus VP 2 base
The primer pair and SpeI, SacI and StuI restriction enzyme of cause, it is described to be used to expand chicken infectivity bursa of Fabricius virus
The sequence of the primer pair of VP2 genes is as follows：
PAU：5’ATCTTGGGTATGTGAGGCTG 3’
PAD：5’TATGGCCCGGATTATGTCTT 3’.
In described kit, it is preferred that also comprising rTaq enzymes, 10 × PCR buffer solutions, dNTP, RNase inhibitor, anti-
Transcriptase, 5 × RT buffer solutions and enzyme cutting buffering liquid.
Described kit is used to carry out in accordance with the following methods when differentiating chicken infectivity bursa of Fabricius virus highly-wetting liquid：
(1) RNA extraction and reverse transcription is carried out to chicken infectivity bursa of Fabricius virus to be detected, obtains virus
cDNA；
(2) PCR is expanded；
The cDNA obtained using step (1) is template, using PAU, PAD as upstream and downstream primer, the mesh in PCR amplification VP2 sequences
Genetic fragment, reaction system is as follows：μ L of cDNA 4, μ L of rTaq enzymes 0.5, the μ L of 10 × PCR buffer solutions 2.5, dNTP 3 μ L, primer
μ L of PAU 1, μ L of primer PAD 1, μ L of deionized water 13, the μ L of cumulative volume 25；
Wink is from being put into PCR instrument, amplification program is as follows after sample blending：95℃5min；94 DEG C of 30sec, 57.1 DEG C
45sec, 72 DEG C of 50sec, 30 circulations；72℃10min；4 DEG C of preservations；
(3) recovery and purifying of viral objective gene sequence PCR primer
The PCR primer of step (2) is subjected to purifying recovery, the DNA being collected into is stored in -20 DEG C；
(4) RFLP identifies IBDV strains
The PCR purified products obtained respectively to step (3) with tri- kinds of restriction enzymes of SpeI, SacI and StuI carry out enzyme
Cut identification；Digestion system is as follows：10U/uL SpeI or StuI or μ L of SacI 1, μ L of enzyme cutting buffering liquid 2,20~40ng/ μ L
μ L of PCR purified products 7, the μ L of deionized water 10, the μ L of cumulative volume 20；
Mixing is put into 37 DEG C of water-baths after water-bath 2h, is mixed with 10 × Loading of 2uL Buffer, with 0.8% agar
Sugared gel observes digestion result through 110V electrophoresis 20min；
(5) result judgement
If being cut out 531bp and 302bp two sections of fragments by SpeI, 242bp and 591bp two sections of fragments are cut out by StuI,
And highly virulent strain can not be then determined as by SacI digestions；
If only being cut out 218bp and 615bp two sections of fragments by SacI, and can not then be sentenced by SpeI and StuI digestions
It is set to low virulent strain.
Further, the invention also provides described kit is strong in preparation discriminating chicken infectivity bursa of Fabricius virus
Purposes in low virulent strain reagent.
The differential diagnostic method based on RT-PCR and RFLP technologies that the present invention is established can distinguish IBDV virulent
Strain and low virulent strain, and step is simply easy to operation, and a kind of new method is provided for IBDV rapid differential diagnosis.
Brief description of the drawings
Fig. 1 is sequence analysis and the selection of restriction enzyme site；
Wherein, the square enclosed represent respectively the SacI restriction enzyme sites of nucleotide sequence the 862nd~867, the 888th~
The StuI restriction enzyme sites of 893 and the SpeI restriction enzyme sites of 1179~1184；
Fig. 2 is the PCR amplifications of viruses indentification；
M：DNA Marker DL2000；1~15：HR、CF、SD、DB11、BC、DN-04、CA、C-8、05-6、99-3、JC-
7th, BD, CEF94, SD-F3 and GC-7 strain pcr amplification product；16：Water compares；
Fig. 3 is the PCR amplifications of the objective gene sequence for RFLP authentication methods；
M：DNA Marker DL2000；1~15：HR、CF、SD、DB11、BC、DN-04、CA、C-8、05-6、99-3、JC-
7th, BD, CEF94, SD-F3 and GC-7 strain pcr amplification product；16：Water compares；
Fig. 4 is HR, CF, SD, DB11, BC and DN-04 strain digestion qualification result；
M：DNA Marker DL2000；1：Cut through SpeI is mono-；2：Cut through SacI is mono-；3：Cut through StuI is mono-；
Fig. 5 is CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strain digestion qualification result；
M：DNA Marker DL2000；1：Cut through SpeI is mono-；2：Cut through SacI is mono-；3：Cut through StuI is mono-；
Fig. 6 is hemagglutination test result；
Fig. 7 is that infected group attacks the bursa of farbricius and spleen after poison and observes lesion；
Fig. 8 is leg muscle bleeding；
Fig. 9 is that infected group observes Pathologic changes with the control group bursa of farbricius and spleen；
From left to right, C represents PBS control group organ, is followed successively by the organ and 9 kinds of weak malicious devices for attacking 6 kinds of virulents afterwards
Official；
Figure 10 is PBS control group chicken bursa and spleen pathologic examination (40 ×)；
A.PBS control group chicken bursas；B.PBS control group chicken spleens；
Figure 11 is bursa of farbricius Histopathologic changes (40 ×) after infection；
A-O infects HR, CF, SD, DB11, BC, DN-04, CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD- respectively
F3 and GC-7 strains；
Figure 12 is spleen Histopathologic changes (40 ×) after infection.
A-O infects HR, CF, SD, DB11, BC, DN-04, CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD- respectively
F3 and GC-7 strains.
Embodiment
Below by embodiment the present invention is described further checking, all embodiments be only used for illustration the present invention,
Do not limit the scope of the invention.For the change done in the claims in the present invention or equivalent change, the present invention is each fallen within
Protection domain within.
Material and its source involved by the present embodiment：
1 experimental animal
10 age in days SPF chicken embryos, 40 age in days SPF chickens, buy in Harbin Veterinary Medicine Inst., China Academy of Agriculture.
2 viruses
IBDV CEF94 strains and CA strains are preserved by this laboratory to be provided；IBDV HR、CF、SD、BC、DB11、DN-04、C-8、
05-6,99-3, JC-7, BD, SD-F3 and GC-7 strain are separated by this laboratory and preserved.
3 kits
QIAquick Gel Extraction Kit, purchased from QINGEN companies；GoTaq qPCR Master Mix, are purchased from
Promega companies.
4 toolenzymes and related reagent
RTaq enzymes, restriction enzyme SpeI, StuI and SacI, RNase inhibitor (RRI), reverse transcriptase RT Ace,
DNA Marker, universal primer Oligo (dT) are purchased from Dalian treasured biotech firm；96 hole V-type blood-coagulation-boards, purchased from Wuxi sky Kanggong
Department.
The preparation of 5 common solvents
PBS：NaCl 4.0g, KCl 0.1g, KH2PO40.1g, Na2HPO4·12H2O 1.45g, ultra-pure water are fixed
Hold to 500mL, 4 DEG C of preservations after sterilizing.
TAE buffer solutions (50 ×)：Tris-Base 121.14g, glacial acetic acid 28.55mL, EDTA 9.3g, ultra-pure water constant volume
To 500mL, pH to 8.0 is adjusted, 4 DEG C save backup.
Alsever's Solution：Dextrose 1.025g, sodium citrate 0.4g, NaCl 0.21g, citric acid 0.028g, incorporate 50mL
4 DEG C of refrigerators are put into ultra-pure water, after autoclaving to save backup.
1% chicken erythrocyte suspension：Syringe draws Alsever's Solution, gathers chicken blood and mixes (ratio 1：4 or so).Add about
The PBS of ten times of blood volumes, slowly overturning makes blood be mixed with PBS, 2000rpm centrifugation 10min, abandoning supernatant, draws the white of top layer
Cell simultaneously discards, and is repeated several times until supernatant is as clear as crystal.The erythroprecipitin of 1 volume is taken to be entered with the PBS of 9 volumes
Row dilution, is configured to 10% chicken red blood cell after shaking up, then take the 10% red blood cell addition 9 volume PBS progress of 1 volume dilute again
Release, it is standby that 4 DEG C of refrigerators are put into after mixing.
6 key instruments and experimental facilities
Grads PCR instrument：Eppendoff Mustercycler Grudient PE2400；
Low temperature table model high speed centrifuge：Beckman AvantiTM 30；
Superclean bench：Froma Scientific 1829.SN.17069-15；
Electrophoresis apparatus：Bio Rad MA120 type MINI VERITICAL GEL SYSTEM；
Horizontal electrophoresis tank DYC-31A types：Liuyi Instruments Plant, Beijing；
Gel imager：UVR-800UK；
Micro electronic balance：METTER AE260 types, Deltalange companies；
M9B88 micro-wave ovens：South Korea's Samsung；
Pure water meter：The types of Millipore Milli-Q II；
Micropipettor：Gilson Inc；
Constant temperature gas bath shaking table：HWY-100B types, Taicang person of outstanding talent's honesty test Instrument Ltd.；
Vortex oscillator：H-1 types, Shanghai Luxi instrument plant；
- 20 DEG C of deep freezers, -4 DEG C of refrigerators：Company of Haier；
- 80 DEG C of ultra low temperature freezers：Revco companies；
Damp and hot high-pressure sterilizing pot：Japanese Sanyo；
Desk centrifuge：The analytical instrument factory of Shanghai the 3rd；
Ultraviolet specrophotometer：Shimadzu UV-120-02；
Biohazard Safety Equipment：Beijing Dong Lianhaer Instrument Ltd.；
The quantitative fluorescent PCRs of ABI PRISM 7500：American AB I companies.
Embodiment 1RT-PCR combinations RFLP detects the foundation of IBDV methods
1st, method
1.1 sequence analyses and the selection of restriction enzyme site
Altogether choose 14 plants known to virulence IBDV strains carry out sequence analysis, respectively highly virulent strain OKYM, D6948, IM,
KKI, KSH, SH95, Gx and UK661 strain, and low virulent strain ZJ2000, B87, CEF94, D78, HZ-2 and JD-1 strain, selected strain
Nucleotide sequence have been subjected to numerous studies and be embodied in ncbi database, strain essential information refers to table 1.Pass through
DNAMAN softwares compare the nucleotide sequence of above-mentioned 14 plants of strains, determine that IBDV highly virulent strains and the difference of low virulent strain sequence are main
Concentrate in the important Viral structural protein VP2 gene regions of IBDV, pass through NEBcutter software analysis highly-wetting liquid sequence difference collection
The specific cleavage site at base mutation position in middle region, tri- kinds of SpeI, SacI and StuI is finally selected as differentiation IBDV
The restriction enzyme of highly-wetting liquid.
The source of 1 14 plants of IBDV strains of table and information
Type：The weak poison of VV=virulents ATT=
The design and synthesis of 1.2PCR primers
With reference to the common conservative regions of VP2 of above-mentioned 14 plants of IBDV highly-wetting liquids in GenBank, Primer Analysis Software is utilized
Oligo 6.0 designs pair of primers PAU, PAD, and primer sequence refers to table 2.This it is amplifiable to primer go out IBDV powers virulence it is different
The target gene fragment of 833bp in the VP2 genes of strain, this section of gene include the main concentration zones of highly-wetting liquid base mutation
The gene order in domain；Three kinds of different restriction enzyme SpeI, SacI and StuI that the gene can be used in RFLP methods simultaneously
Carry out digestion identification IBDV strains.
Meanwhile to identify the IBDV strains of this laboratory preservation, the IBDV that synthetic world animal health tissue (OIE) is recommended
Primer L2, U2 of strain RT-PCR detection method, primer sequence refer to table 2.This is designed according to VP2 gene orders to primer,
The expection size of PCR primer is 604bp.
Real-time fluorescence quantitative PCR primer S1, S2 for Viral Quantification include VP4 according to the design of VP4 conserved genetic sequences
One section of sequence in gene order in 302bp~540bp.
Above primer synthesizes by SBS Genetech gene technology Co., Ltd.
The primer sequence of table 2
The extraction and reverse transcription of 1.3 viral RNAs
Take out -80 DEG C of refrigerators in this laboratory preserve 15 plants of IBDV strains, respectively CEF94, BD, CA, HR, CF, SD,
BC, DB11, DN-04, C-8,05-6,99-3, JC-7, SD-F3 and GC-7, the extraction and reverse transcription experiment of viral RNA are carried out,
Method detailed is as follows：
(1) virus liquid 250uL is respectively taken to be extracted after being put into 1.5mL EP pipes.
(2) 500uL Trizol are added into EP pipes quickly to spin upside down, shake 2min, be placed in 10min on ice, centre shake
Swing 1min.
(3) separation phase：After addition 350uL chloroforms that EP lids is tight, acutely concussion is well mixed wherein liquid, on ice
After standing 10min, 12000rpm centrifugation 7min, after centrifugation terminates, stratification state is presented in liquid in pipe, and the bottom is red benzene
Phenol-chloroform layer, center are " tunica albuginea " layers, and the top is transparent water sample layer, and RNA is all stayed in water sample layer.
(4) RNA precipitation：Slowly supernatant liquid is moved into the clean 1.5mL EP pipes without RNase (about
400~500uL), the isopropanol of equal volume is added, is put into after mixing in -20 DEG C and is more than 1h and then 12000rpm centrifugations 7min.
(5) RNA washing：Supernatant carefully is outwelled, leaves and takes precipitation.Add 75% absolute ethyl alcohol (the DEPC water that 1mL now matches somebody with somebody
Prepare, precooling) wash RNA precipitate once, then 12000rpm centrifuges 7min.
(6) RNA redissolution：Supernatant carefully is outwelled, by pipe back-off on blotting paper, the naturally dry in workbench, no
RNA precipitate can be allowed to be completely dried.22uL DEPC water dissolving is added into pipe again, total serum IgE is obtained, is inverted immediately afterwards
Record.
(7) 2uL universal primers Oligo (dT), 75 DEG C of water-bath 10min, then ice bath 5min are added.
(8) RRI 2uL, RT Ace 2uL, 5 × RT Buffer 8uL and dNTP 4uL are added in EP pipes, altogether 40uL bodies
System mixes.
(9) it is suitable to be managed from EP, cDNA is obtained after 42 DEG C of water-bath 1h 30min, 72 DEG C of 10min, ice bath 5min, -20 DEG C of preservations are standby
With.
1.4PCR amplification
(1) the PCR amplifications of viruses indentification
Using the cDNA obtained in 1.3 experiments as template, L2, U2 are upstream and downstream primer, enter performing PCR amplification with identification experiment room
Preserve 15 plants of strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8,05-6,99-3, JC-7, SD-F3 and
GC-7 strains, reaction system are as follows：
Wink is from being put into PCR instrument, amplification program after sample blending：94℃5min；94 DEG C of 30sec, 56 DEG C of 45sec, 72 DEG C
1min, 35 circulations；72℃10min；4 DEG C of preservations.
After PCR reactions terminate, PCR primer and 2.5uL10 × Loading Buffer are mixed, with DNA Marker
DL2000 is control, with 0.8% Ago-Gel through 110V electrophoresis 20min, observes PCR amplification.
(2) PCR for the objective gene sequence of RFLP authentication methods is expanded
Using the cDNA obtained in 1.3 experiments as template, PAU, PAD are upstream and downstream primer, and PCR expands the mesh in VP2 sequences
Genetic fragment, reaction system is as follows：
Wink is from being put into PCR instrument, amplification program is as follows after sample blending：95℃5min；94 DEG C of 30sec, 57.1 DEG C
45sec, 72 DEG C of 50sec, 30 circulations；72℃10min；4 DEG C of preservations.
After PCR reactions terminate, PCR primer and 2.5uL10 × Loading Buffer are mixed, with DNA Marker
DL2000 is control, with 0.8% Ago-Gel through 110V electrophoresis 20min, observes PCR amplification.
The recovery and purifying of 1.5 viral objective gene sequence PCR primers
10 × Loading of above-mentioned PCR primer 50uL and 5uL Buffer are uniformly mixed together, carry out 1% agar
Sugared gel electrophoresis, using DNA Marker DL2000 as control, after electrophoresis terminates, using QIAquick Gel Extraction
Kit recovery purifying PCR primers.Operated by kit specification.The DNA being collected into is stored in -20 DEG C and is used for subsequent experimental.
The sequencing of 1.6 purified pcr products
SBS Genetech gene skill will be sent to primer L2, U2 and PAU, 15 strain virus of PAD amplifications PCR primer after purification
Art Co., Ltd is sequenced.
1.7RFLP identifies IBDV strains
It is used for RFLP to what is expanded through primer PAU, PAD respectively with tri- kinds of restriction enzymes of SpeI, SacI and StuI
The PCR purified products of the objective gene sequence of authentication method carry out digestion identification.Digestion system is as follows：
Mixing is put into 37 DEG C of water-baths after water-bath 2h, is mixed with 10 × Loading of 2uL Buffer, with 0.8% agar
Sugared gel observes digestion result through 110V electrophoresis 20min.
2 results
The selection of 2.1VP2 gene sequencings and restriction enzyme site
Found after Multiple Sequence Alignment, the different IBDV strains of selected 14 plants of virulence in VP2 gene orders
The region that one section of length is 833bp between 521bp~1354bp be present, the base mutation of highly-wetting liquid is concentrated mainly on the region.
Low virulent strain ZJ2000, B87, CEF94, D78, HZ-2 and JD-1 have in this section of region at 862~867bp nucleotide positions
Have common restriction enzyme site SacI, a sequence GAGCTC, but other 8 plants of highly virulent strain D6948, Gx, IM, KKI, KSH,
OKYM, SH95 and UK661 do not have SacI restriction enzyme sites in identical opening position, because base ' G ' is taken by ' A '
Generation, i.e. GAACTC.8 plants of highly virulent strains restrictive restriction enzyme site at 888~893bp nucleotide positions in this region
StuI, sequence AGGCCT；Restrictive restriction enzyme site SpeI, sequence are at 1179~1184bp nucleotide positions
ACTAGT.And 6 plants of low virulent strains in identical opening position without two kinds of restriction enzyme sites of StuI and SpeI because in 888bp
Locate base ' A ' by ' T ' to be substituted, base ' A ' is by ' G ' substitution (such as Fig. 1) at 1179bp.Therefore, in this section of 833bp sequence area
Interior, highly virulent strain can be cut out 531bp and 302bp two sections of fragments by restriction enzyme SpeI, can also be cut out by StuI
242bp and 591bp two sections of fragments, and can not be by SacI digestions；Low virulent strain only by SacI digestions, cuts out 218bp and 615bp
Two sections of fragments.Therefore, choose restriction enzyme SacI, StuI and SpeI as 521bp in digestion VP2 gene orders~
A length of 833bp genetic fragment between 1354bp, to establish the RT-PCR combination RFLP antidiastoles for distinguishing IBDV highly-wetting liquids
Method.
The PCR amplifications of 2.2 viruses indentifications
15 plants of IBDV strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8,05- that laboratory is preserved
6th, 99-3, JC-7, SD-F3 and GC-7 carry out RNA extraction and reverse transcription, and the cDNA obtained using reverse transcription is template, with OIE
Primer L2, U2 of recommendation carry out the PCR amplifications of viruses indentification, and as a result visible 604bp purpose band, is consistent with expection, sequence
Measurement result is correct, illustrates that 15 plants of strains that laboratory preserves are IBDV strains.As shown in Figure 2.
2.3 are used for the PCR amplifications of the objective gene sequence of RFLP authentication methods
15 plants of IBDV strains CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8,05- that laboratory is preserved
6th, 99-3, JC-7, SD-F3 and GC-7 carry out RNA extraction and reverse transcription, using the cDNA of acquisition as template, with primer PAU,
PAD carries out the PCR amplifications for the objective gene sequence of RFLP authentication methods, as a result visible 833bp purpose band, with expection
It is consistent, as a result such as Fig. 3.
The sequencing results of 2.4 purified pcr products
As shown in SEQ ID NO.1-15, sequencing result shows the PCR primer sequencing result of 15 strain virus of PAU, PAD amplification
Show, highly virulent strain is respectively provided with StuI and SpeI restriction enzyme sites, and low virulent strain is respectively provided with SacI restriction enzyme sites, and sequencing result is correct.
2.5RFLP identifies IBDV strains
SpeI, SacI and StuI are used respectively after PCR primer for the objective gene sequence of RFLP authentication methods is purified
Three kinds of restriction enzymes carry out digestion, as a result show：HR, CF, SD, DB11, BC and DN-04 strain can be cut out by SpeI
Two sections of fragments of 531bp and 302bp sizes, two sections of fragments of 242bp and 591bp sizes can also be cut out by StuI, but can not
Cut (such as Fig. 4) by SacI；CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strain can only be cut by SacI
Go out two sections of fragments of 218bp and 615bp sizes, and (such as Fig. 5) can not be cut by SpeI and StuI.Show HR, CF, SD, DB11,
BC and DN-04 strains are IBDV highly virulent strains, and CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strain are equal
For IBDV low virulent strains.
The animal of embodiment 2 returns experimental identification IBDV strain virulence
1st, method
The rejuvenation of 1.1 viruses
Taken out from -80 DEG C of refrigerators this laboratory preservation 15 plants of IBDV strains CEF94, BD, CA, HR, CF, SD, BC,
DB11, DN-04, C-8,05-6,99-3, JC-7, SD-F3 and GC-7,7000rpm4 DEG C of centrifugation 10min, use disposable syringe
Supernatant is drawn, virus sterilizing PBS 100 is diluted, dilution allantocherion vaccination is in 10 age in days SPF chicken embryos, every kind of disease
Malicious dilution is inoculated with two chicken embryos.Each egg inoculation about 200uL virus liquids.Chicken embryo paraffin sealing after inoculation, then puts
It is incubated in 37 DEG C of incubators.Chicken embryo dead in 24h is discarded, examines embryo twice daily, dead chicken embryo is taken out at any time, until 96h
All chicken embryos are together taken out, chicken embryo is placed in 4 DEG C of refrigerator 4h so as to carry out subsequent experimental.
The chicken embryo of inoculation is taken out from 4 DEG C of refrigerators and is placed into Biohazard Safety Equipment, it is artificial to open chicken embryo with sterile tweezers
The eggshell at air chamber position, allantoic fluid is drawn in sterile chamber with the syringe of disposable needle-less, while collect lesion fetus
And chorioallantoic membrane, lesion fetus and chorioallantoic membrane are placed in mortar, pours into after liquid nitrogen to be ground with pestle and is mixed with allantoic fluid, 5000rpm
4 DEG C of centrifugation 20min, collection supernatant are placed on -80 DEG C of refrigerators and saved backup.
The degree of purity of 1.2 blood coagulation tests identification rejuvenation virus
(1) in 96 hole V-type blood-coagulation-boards, 25uL PBS are added per hole.
(2) 25uL virus liquids are added into the first hole, 25uL are suctioned out after mixing into the second hole, according to said method multiple proportions successively
The 11st hole is diluted to, finally discards 25uL liquid, the 12nd hole compares for red blood cell.
(3) 25uL PBS are added to every hole successively.
(4) chicken erythrocyte suspensions of 25ul 1% are added to every hole successively.
(5) 2min will be shaken on blood-coagulation-board earthquake device, is stored at room temperature 30min, when the red blood cell of control wells is significantly into knob
Result of determination when buckle-like.
(6) result judgement：Blood-coagulation-board is tilted into certain angle, recognized if red blood cell is deposited on bottom hole and is glided in teardrop shaped
It is set to without coagulation.If red blood cell is laid in bottom hole, teardrop shaped do not occur for red blood cell glided then to have regarded as after blood-coagulation-board tilts
It is coagulation.
If virus to be measured without coagulation, shows the just viscous and paramyxovirus without birds in the virus, then the sample is judged
For pure IBDV, and without other common disease poultry and livestock poison mixed infections.
1.3 real time fluorescence quantifying PCR methods detect each strain virus copy number
Experimental method according to 1.3 sections of embodiment 1 extracts the RNA of 15 strain virus after rejuvenation and carries out reverse transcription acquisition
CDNA, using this cDNA as template, S1, S2 are respectively as upstream and downstream primer (table 1 of embodiment 1), according to GoTaq qPCR
Master Mix explanations carry out real-time PCR, and IBDV calibration curve equation y=- has been established according to this laboratory
3.482x+51.696 calculating the copy number of this 15 strain virus, amplification system is as follows：
Wink is from being put into amplification program in instrument after sample blending：95℃2min；95 DEG C of 15sec, 60 DEG C of 1min, 40 circulations；
95℃15sec；60℃15sec；95℃15sec.
1.4 zoogenetic infections are tested
Viral copy number according to calculating is adjusted for the 15 strain virus dosage for attacking poison into identical copy number, will be quantitative
Virus afterwards is diluted with sterilizing PBS, and carrying out eye droppings to 40 age in days SPF chickens attacks poison, and it is every 100uL to attack poison and measure, control group
Eye droppings is carried out with the PBS of same volume.Each group shares 6 SPF chickens.Attack and observe and record each group chicken after poison at any time and shown
Clinical symptoms, after infection the 3rd day every group randomly select 3 chickens and cut open killing, record observing of showing after each group chicken cut open inspection
Lesion.The bursa of farbricius and spleen are taken, carries out making and the pathologic examination of paraffin section.
The making of 1.5 pathological sections
(1) fast flushing is repaiied：Fixed tissue repair soon, size is about 1.0cm × 1.0cm × 0.5cm, and flowing water rushes
Wash, rinsed overnight if the tissue block set time is longer；
(2) it is dehydrated：The tissue block rinsed is taken out, carries out tissue block dehydration, sequentially stays overnight -80% ethanol for 70% ethanol
II 20min of the ethanol of 20min -100% of the ethanol of 1.5h -95% 15min -100% ethanol I-acetone 10min, is careful not to take off
Water is excessive；
(3) it is transparent：First have to carry out the transparent of tissue block after dehydration, be sequentially I 10min of dimethylbenzene-dimethylbenzene II
15min, soak 1h in 60 DEG C of soft waxs；
(4) waxdip：Soft wax is poured into ready-made small paper groove, is quickly put into tissue block wherein, pay attention between tissue away from
From wanting reasonable, place more than 2h and make soft wax solidification abundant；
(5) embed：Embedded small wax stone is taken out, wax stone is accomplished into suitable size with pocket knife, it is finally that wax stone is careful
Fixed on wood particle；
(6) cut into slices：It is noted that the thickness of section, thickness are advisable in 5um, extended piece in constant water bath box during section,
Temperature is 45 DEG C；Finally the slice, thin piece cut is placed in 37 DEG C of incubators and bakes piece；
(7) dewax：The order of dewaxing is the second of 3min -95% of the 5min -95% of I 5min of dimethylbenzene-dimethylbenzene II ethanol I
The ethanol of 3min -80% ethanol of 3min -70% of alcohol II 3min-distilled water 10min；
(8) dye：It is sequentially haematoxylin 7min-distillation according to the new and old adjustment haematoxylin dyeing time of dye liquor during dyeing
Water a moment, 1% hydrochloride alcohol differentiation 20s-flowing water rinse 2h-eosin stains 8min-distilled water a moment；
(9) it is dehydrated：Order for the 10min of 70% ethanol of ethanol 20s -80% ethanol of 20s -95% 5min -100% second I -
The 5min of 100% ethanol II；
(10) transparent and mounting：Order for the 5min of I 10min of dimethylbenzene-dimethylbenzene II-gummy mounting -37 DEG C drying -
Microscopy.
2nd, result
The rejuvenation of 2.1 viruses
By 10 age in days SPF chick chorioallantoic membranes be inoculated with, to this laboratory preserve 15 plants of IBDV strains BD, CA, HR,
CF, SD, BC, DB11, DN-04, CEF94, C-8,05-6,99-3, JC-7, SD-F3 and GC-7 strain carry out the rejuvenation of virus.Collect
The fetus of allantoic fluid, chorioallantoic membrane and lesion, chorioallantoic membrane and fetus are ground by liquid nitrogen and mixed with allantoic fluid, centrifuging and taking supernatant,
The virus as harvested, it is placed in -80 ° of refrigerators and saves backup.
The degree of purity of 2.2 blood coagulation tests identification rejuvenation virus
15 plants of IBDV strains that laboratory preserves carry out blood coagulation tests after poison is received in rejuvenation, as a result show and blood-coagulation-board inclines
Red blood cell is deposited on bottom hole and glided (such as Fig. 6) in teardrop shaped after oblique certain angle, therefore without coagulation, then judgement experiment
CEF94, BD, CA, HR, CF, SD, BC, DB11, DN-04, C-8,05-6,99-3, JC-7, SD-F3 and GC-7 totally 15 that room preserves
Strain strain is pure IBDV, and without other common disease poultry and livestock poison mixed infections.
2.3 real-time fluorescent quantitative RT-PCR methods detect each strain virus copy number results
15 strain virus are carried out to RNA extraction and reverse transcription after chicken embryo rejuvenation, using the cDNA of acquisition as template, S1,
S2 carries out real-time PCR respectively as upstream and downstream primer according to GoTaq qPCR Master Mix.
According to laboratory preserve 15 plants of IBDV strains amplification curve can obtain CEF94, BD, CA, HR, CF, SD, BC,
DB11, DN-04, C-8,05-6,99-3, JC-7, SD-F3 and GC-7 strain reach the averaging loop required for fluorescent absorption threshold value
Number, be followed successively by 19.876,24.722,30.389,30.914,28.458,29.279,31.886,31.425,31.977,
29.688、28.043、26.953、23.957、21.240、23.139.IBDV standard curve side has been established according to this laboratory
Journey y=-3.482x+51.696 can obtain copies=10Thus the copy number of each strain virus is calculated, is tied
Fruit is as shown in table 3：
The copy number of 3 15 plants of strains of table
Clinical symptoms and pathological anatomy change after 2.4 zoogenetic infections
Attack 48h after poison, each group chicken of infection HR, CF, SD, DB11, BC and DN-04 strain virus show spirit it is depressed,
Feed intake is reduced, feather is in disorder, head and wing are sagging, a dispirited, slow-witted vertical a corner and catacleisis, and the clinical condition such as be reluctant to walk about
Shape；PBS control group chicken is acted normally, and the chicken of other weak poison groups is without obvious clinical symptoms.
The 3rd day after infection, every group randomly selects 3 chickens and cut open killing, each group chicken through cut open inspection 15 strain virus of visible infection
Different degrees of characteristic lesion occurs for the bursa of farbricius, wherein the weaker poison group of lesion of observing of poison group chicken wants obvious by force, such as Fa Shi
Yellow gel-shaped oedema is presented in capsule, covered with cream-colored fibrinous exudate (as shown in Figure 7 A) on mucous membrane；Spleen enlargement, it is individual
There is not bleeding (as shown in Figure 7 B)；There is striated bleeding (such as Fig. 8) in muscle on the inside of the Flaccid Coelogyne of the strong poison of infection.Infect weak
Also there is the phenomenon of the bursa of farbricius and spleen enlargement in the chicken of poison group, but is contrasted with virulent group, and situation is relatively light；PBS control
Histoorgan does not have obvious Pathologic changes (such as Fig. 9) after group chicken cut open inspection.
Histopathologic changes after 2.5 zoogenetic infections
The bursa of farbricius and spleen are taken, carries out the making of paraffin section, the PBS control group bursa of farbricius and spleen tissue structure tend to just
Often, obvious Histopathologic changes (such as Figure 10) are not observed；6 plants of highly virulent strain HR, CF, SD, DB11, BC and DN-04 strains exist
Attack bursa of farbricius pathologic examination after poison, it is seen that substantial amounts of lymphocyte necrosis and with apoptosis phenomenon, have in lymph follicle
Substantial amounts of different preferendum cellular infiltration, visible vacuolization in the folliculus of part, lamina propria oedema, there is inflammatory cell infiltration in lamina propria
(such as Figure 11 A-F).9 plants of low virulent strain CA, C-8,05-6,99-3, JC-7, BD, CEF94, SD-F3 and GC-7 strain Fa Shi after poison is attacked
Capsule pathologic examination, it is seen that lymph follicle atrophy, lymphocyte therein substantially reduce, and have inflammatory cells in lamina propria
Infiltrate (such as Figure 11 G-O).
Laboratory preserve 15 plants of strain HR, CF, SD, DB11, BC, DN-04, CA, C-8,05-6,99-3, JC-7, BD,
CEF94, SD-F3 and GC-7 spleen pathologic examination after poison is attacked, significant difference is had no between highly virulent strain and low virulent strain,
It can be seen that lymphocyte significantly reduces, and white space (such as Figure 12 A-O) is formed with substantial amounts of macrophage hyperplasia.
Sequence table
<110>Northeast Agricultural University
<120>The kit of chicken infectivity bursa of Fabricius virus highly-wetting liquid is differentiated based on RT-PCR and RFLP technologies and its answered
With
<130> KLPI170687
<160> 15
<170> PatentIn 3.5
<210> 1
<211> 773
<212> DNA
<213>IBDV HR strains
<400> 1
cccaaaatgg tagcaacatg tgacagcagt gacaggccca gagtctacac cataactgca 60
gccgatgatt accaattctc atcacagtac caaacaggtg gagtaacaat cacactgttc 120
tcagctaata tcgatgccat cacaagcctc agcatcgggg gagaactcgt gtttcaaaca 180
agcgtccaag gccttatact gggtgctacc atctacctta taggctttga tgggactgcg 240
gtaatcacca gagctgtggc tgcagacaat gggctaacgg ccggcactga caaccttatg 300
ccattcaata ttgtgattcc aaccagcgag ataacccagc caatcacatc catcaaacta 360
gagatagtga cctccaaaag tggtggtcag gcgggagatc agatgtcatg gtcagcaagt 420
gggagcctag cagtgacgat ccacggtggc aactatccag gggccctccg tcccgtcaca 480
ctagtagcct acgaaagagt ggcaacagga tctgtcgtta cggtcgccgg ggtgagcaac 540
ttcgagctga tcccaaatcc tgaactagca aagaacctgg tcacagaata cggccgattt 600
gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660
aagaccgtct ggccaacaag ggagtacact gactttcgcg agtacttcat ggaggtggcc 720
gacctcaact ctcccctgaa gattgcagga gcattcggct tcaaagctcc ccg 773
<210> 2
<211> 833
<212> DNA
<213>IBDV CF strains
<400> 2
cggtcgccgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60
tcacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120
agagggaccg tcttggcatc aagaccgtat ggccaacaag ggagtacact gactttcgcg 180
agtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga gcatttggct 240
tcaaagacat aatccgggcc ctaaggagga tagctgtgcc ggtggtctct acactgttcc 300
cacccgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360
aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420
gccgcataag gcagctaact ctcgccgccg acaaggggta cgaggtagtc gcgaatctgt 480
tccaggtgcc ccagaatcct gtagtcgacg ggattctcgc ttcacccggg atactccgcg 540
gtgcacacaa cctcgactgc gtgttgagag agggtgccac gctattccct gtggtcatca 600
cgacagtgga agatgccatg acacccaaag cactgaacag caaaatgttt gctgtcattg 660
aaggcgtgcg agaagatctc caacctccat ctcaaagagg atccttcata cgaactctct 720
ccggacatag agtctatgga tatgctccag atggggtact tccactggag actgggagag 780
tttacaccgt ggtcccaata gatggtgtct gggacgacag cattatgctg tcc 833
<210> 3
<211> 773
<212> DNA
<213>IBDV SD strains
<400> 3
cccaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60
gccgatgatt accaattctc atcacagtac caatcaggtg gggtaacaat cacactgttc 120
tcagccaaca ttgatgctat cacaagcctc agcattgggg gagaactcgt gttccataca 180
agcgtccaag gccttgcact gaacgccacc atctacctta taggctttga tgggactaca 240
gtaatcacca gagctgtggc ctcagacaat gggctgacta ccggcatcga caatcttatg 300
ccattcaatc ttgtgattcc aaccaacgag ataacccagc caatcacatc catcaaactg 360
gagatagtga cctccaaaag tggtggtcag gcaggggacc agatgtcatg gtcggcaagt 420
gggagcctag cagtgacaat ccatggtggc aactatccag gggccctccg tcccgtcaca 480
ctagtagcct acgaaagagt ggcaacagga tccgtcgtta cggtcgccgg ggtgagcaac 540
ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600
gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660
aagaccgtct ggccaacaag ggagtacact gactttcgtg agtacttcat ggaggtggcc 720
gacctcaact ctcccctgaa gattgcagga gcatttggct tcaaagctcc cgg 773
<210> 4
<211> 773
<212> DNA
<213>IBDV DB11 strains
<400> 4
cccaaaaatg gtagccacat gtgacagcag tgacaggccc agagtctaca ccataactgc 60
agccgatgat taccaattct catcacagta ccaatcaggt ggggtaacaa tcacactgtt 120
ctcagccaac attgatgcta tcacaagcct cagcattggg ggagaactcg tgttccatac 180
aagcgtccaa ggccttgcac tgaacgccac catctacctt ataggctttg atgggactac 240
agtaatcacc agagctgtgg cctcagacaa tgggctgact accggcatcg acaatcttat 300
gccattcaat cttgtgattc caaccaacga gataacccag ccaatcacat ccatcaaact 360
ggagatagtg acctccaaaa gtggcggtca ggcaggggac cagatgtcat ggtcggcaag 420
tgggagccta gcagtgacaa tccatggtgg caactatcca ggggccctcc gtcccgtcac 480
actagtagcc tacgaaagag tggcaacagg atccgtcgtt acggtcgccg gggtgagcaa 540
cttcgagctg atcccaaatc ctgaactagc aaagaacctg gttacagaat acggccgatt 600
tgacccagga gccatgaact acacaaaatt gatactgagt gagagggacc gtcttggcat 660
caagaccgtc tggccaacaa gggagtacac tgactttcgt gagtacttca tggaggtggc 720
cgacctcaac tctcccctga agattgcagg agcattcggc ttcaagctcc ggg 773
<210> 5
<211> 777
<212> DNA
<213>IBDV BC strains
<400> 5
cccacaaatg gtagccacat gtgacagcag tgacaggccc agagtctaca ccataactgc 60
agccgatgat taccaattct tatcacagta ccaaccaggt ggggtaacaa tcacactgtt 120
ctcagccaac attgatgcta tcacaagcct cagcgttggg ggagaactcg tgtttcaaac 180
aagcgttcaa ggccttgtac tgggcgccac catctacctt ataggctttg atgggactac 240
ggtaatcacc agggctgtgg ccgcagacaa tgggctgacg gccggcaccg acaatcttat 300
gccattcaat attgtgattc caaccaacga gataacccag ccaattacat ccatcaaact 360
ggagatagtg acctccaaaa gtggtggcca ggcaggggac cagatgtcat ggtcggcaag 420
tgggagccta gcagtgacga tccatggtgg caactatcca ggggccctcc gtcccgtcac 480
actagtagcc tacgaaagag tggcaacagg atccgtcgtt acggtcgctg gggtgagcaa 540
cttcgagctg atcccaaatc ctgaactagc aaagaacctg gttacagaat acggccgatt 600
tgacccagga gccatgaact acacaaaatt gatactgagt gagagggacc gtcttggcat 660
caagaccgtc tggccaacaa gggagtacac cgactttcgt gagtacttca tggaggtggc 720
cgacctcaat tctcccctga agattgcagg agcatttggc ttcaaagaca tatcccg 777
<210> 6
<211> 768
<212> DNA
<213>IBDV DN-04 strains
<400> 6
ccaaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60
gccgatgatt accaattctc atcacagtac caatcaggtg gggtaacaat cacactgttc 120
tcagccaaca ttgatgctat cacaagcctc agcattgggg gagaacttgt gttccataca 180
agcgtccaag gccttgcact gaacgccacc atctacctta taggctttga tgggactaca 240
gtaatcacca gagctgtggc ctcagacaat gggctgacta ccggcatcga caatcttatg 300
ccattcaatc ttgtgattcc aaccaacgag ataacccagc caatcacatc catcaaactg 360
gagatagtga cctccaaaag tggcggtcag gcaggggacc agatgtcatg gtcggcaagt 420
gggagcctag cagtgacaat ccatggtggc aactatccag gggccctccg tcccgtcaca 480
ctagtagcct acgaaagagt ggcaacagga tccgtcgtta cggtcgccgg ggtgagcaac 540
ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600
gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660
aagaccgtct ggccaacaag ggagtacact gactttcgtg agtacttcat ggaggtggcc 720
gacctcaact ctcccctgaa gattgcagga gcatttggct tcaagctc 768
<210> 7
<211> 773
<212> DNA
<213>IBDV CA strains
<400> 7
cccaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60
gccgatgatt accaattctc atcacagtac caaccaggtg gggtaacaat cacactgttc 120
tcagccaaca ttgatgccat cacaagcctc agcgttgggg gagagctcgt gtttcaaaca 180
agcgtccacg gccttgtact gggcgccacc atctacctca taggctttgg tgggacagcg 240
gtaatcacca gggctgtggc cgcaaacaat gggctgacga ccggcaccga caaccttttg 300
ccattcaatc ttgtgattcc aacaaacgag ataacccagc caatcacatc catcaaactg 360
gagatagtga cctccaaaag tggtggtcag gcaggggatc agatgtcatg gtccgcaaga 420
gggagcctag cagtgacgat ccatggtggc aactatccag gggccctccg tcccgtcacg 480
ctagtggcct acgaaagagt ggcaacagga tccgtcgtta cggtcgctgg ggtgagcaac 540
ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600
gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660
aagaccgtct ggccaacaag ggagtacact gactttcgtg aatacttcat ggaggtggcc 720
gacctcaact ctcccctgaa gattgcagga gcattcggct tcaaagacta ccc 773
<210> 8
<211> 774
<212> DNA
<213>IBDV C-8 strains
<400> 8
cccaaaaatg gtagccacat gtgacagcag tgacaggccc agagtctaca ccataactgc 60
agccgatgat taccaattct catcacagta ccaaccaggt ggggtaacaa tcacactgtt 120
ctcagccaac attgatgcca tcacaagcct cagcgttggg ggagagctcg tgtttcgaac 180
aagcgtccac ggccttgtac tgggcgccac catctacctc ataggctttg atgggacaac 240
ggtaatcacc agggctgtgg ccgcaaacaa tgggctgacg accggcaccg acaaccttat 300
gccattcaat cttgtgattc caacaaacga gataacccag ccaatcacat ccatcaaact 360
ggagatagtg acctccaaaa gtggtggtca ggcaggggat cagatgtcat ggtcggcaag 420
agggagccta gcagtgacga tccatggtgg caactatcca ggggccctcc gtcccgtcac 480
gctagtggcc tacgaaagag tggcaacagg atccgtcgtt acggtcgctg gggtgagcaa 540
cttcgagctg atcccaaatc ctgaactagc aaagaacctg gttacagaat acggccgatt 600
tgacccagga gccatgaact acacaaaatt gatactgagt gagagggacc gtcttggcat 660
caagaccgtc tggccaacaa gggagtacac tgactttcgt gaatacttca tggaggtggc 720
cgacctcaac tctcccctga agattgcagg agcattcggc ttcaaagact accc 774
<210> 9
<211> 768
<212> DNA
<213>IBDV 05-6 strains
<400> 9
cccaaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60
gccgatgatt accaattctc atcacagtac caaccaggtg gggtaacaat cacactgttc 120
tcagccaaca ttgatgccat cacaagcctc agcgttgggg gagagctcgt gtttcaaaca 180
agcgtccacg gccttgtact gggcgccacc atctacctca taggctttga tgggacagcg 240
gtaatcacca gggctgtggc cgcaaacaat gggctgacga ccggcaccga caacctttag 300
ccattcaatc ttgtgattcc aacaaacgag ataacccagc caatcacatc catcaaactg 360
gagatagtga cctccaaaag tggtggtcag gcaggggatc agatgtcatg gtccgcaaga 420
gggagcctag cagtgacgat ccatggtggc aactatccag gggccctccg tcccgtcacg 480
ctagtggcct acgaaagagt ggcaacagga tccgtcgtta cggtcgctgg ggtgagcaac 540
ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600
gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660
aagaccgtct ggccaacaag ggagtacact gactttcgtg aatacttcat ggaggtggcc 720
gacctcaact ctcccctgaa gattgcagga gcattcggct tcaagctc 768
<210> 10
<211> 768
<212> DNA
<213>IBDV 99-3 strains
<400> 10
atgtgtagcc acatgtgaca gcagtgacag gcccagagtc tacaccataa ctgcagccga 60
tgattaccaa ttctcatcac agtaccaacc aggtggggta acaatcacac tgttctcagc 120
caacattgat gccatcacaa gcctcagcgt tgggggagag ctcgtgtttc aaacaagcgt 180
ccacggcctt gtactgggcg ccaccatcta cctcataggc tttgatggga caacggtaat 240
caccagggct gtggccgcaa acaatgggct gacgaccggc accgacaacc ttatgccatt 300
caatcttgtg attccaacaa acgagataac ccagccaatc acatccatca aactggagat 360
agtgacctcc aaaagtggtg gtcaggcagg ggatcagatg tcatggtcgg caagagggag 420
cctagcagtg acgatccatg gtggcaacta tccaggggcc ctccgtcccg tcacgctagt 480
ggcctacgaa agagtggcaa caggatccgt cgttacggtc gctggggtga gcaacttcga 540
gctgatccca aatcctgaac tagcaaagaa cctggttaca gaatacggcc gatttgaccc 600
aggagccatg aactacacaa aattgatact gagtgagagg gaccgtcttg gcatcaagac 660
cgtctggcca acaagggagt acactgactt tcgtgaatac ttcatggagg tggccgacct 720
caactctccc ctgaagattg caggagcatt cggcttcaaa gactcccg 768
<210> 11
<211> 774
<212> DNA
<213>IBDV JC-7 strains
<400> 11
cgagaaatgg tagccacatg tgacagcagt gacaggccca gagtctacac cataactgca 60
gccgatgatt accaattctc atcacagtac caaccaggtg gggtaacaat cacactgttc 120
tcagccaaca ttgatgccat cacaagcctc agcgttgggg gagagctcgt gtttcaaaca 180
agcgtccacg gccttgtact gggcgccacc atctacctca taggctttga tgggacaacg 240
gtaatcacca gggctgtggc cgcaaacaat gggctgacga ccggcaccga caaccttttg 300
ccattcaatc ttgtgattcc aacaaacgag ataacccagc caatcacatc catcaaactg 360
gagatagtga cctccaaaag tggtggtcag gcaggggatc agatgtcatg gtccgcaaga 420
gggagcctag cagtgacgat ccatggtggc aactatccag gggccctccg tcccgtcacg 480
ctagtggcct acgaaagagt ggcaacagga tccgtcgtta cggtcgctgg ggtgagcaac 540
ttcgagctga tcccaaatcc tgaactagca aagaacctgg ttacagaata cggccgattt 600
gacccaggag ccatgaacta cacaaaattg atactgagtg agagggaccg tcttggcatc 660
aagaccgtct ggccaacaag ggagtacact gactttcgtg aatacttcat ggaggtggcc 720
gacctcaact ctcccctgaa gattgcagga gcattcggct tcaaagacta cccc 774
<210> 12
<211> 833
<212> DNA
<213>IBDV BD strains
<400> 12
gttttgactg gcccttagtc tacaccataa ctgcagccga tgattaccaa ttctcatcac 60
agtaccaacc aggtggggta acaatcacac tgttctcagc caacattgat gccatcacaa 120
gcctcagcgt tgggggagag ctcgtgtttc aaacaagcgt ccacggcctt gtactgggcg 180
ccaccatcta cctcataggc tttgatggga cagcggtaat caccagggct gtggccgcaa 240
acaatgggct gacgaccggc accgacaacc ttttgccatt caatcttgtg attccaacaa 300
acgagataac ccagccaatc acatccatca aactggagat agtgacctcc aaaagtggtg 360
gtcaggcagg ggatcagatg tcatggtccg caagagggag cctagcagtg acgatccatg 420
gtggcaacta tccaggggcc ctccgtcccg tcacgctagt ggcctacgaa agagtggcaa 480
caggatccgt cgttacggtc gctggggtga gcaacttcga gctgatccca aatcctgaac 540
tagcaaagaa cctggttaca gaatacggcc gatttgaccc aggagccatg aactacacaa 600
aattgatact gagtgagagg gaccgtcttg gcatcaagac cgtctggcca acaagggagt 660
acactgactt tcgtgaatac ttcatggagg tggccgacct caactctccc ctgaagattg 720
caggagcatt cggcttcaaa gacatatccc ggggcccata a 761
<210> 13
<211> 833
<212> DNA
<213>IBDV CEF94 strains
<400> 13
cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60
ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120
agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact gactttcgtg 180
aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga gcattcggct 240
tcaaagacat aatccgggcc ataaggagga tagctgtgcc ggtggtctcc acattgttcc 300
cacctgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360
aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420
gccgcataag gcagctgact ctcgccgccg acaaggggta cgaggtagtc gcgaatctat 480
tccaggtgcc ccagaatccc gtagtcgacg ggattcttgc ttcacctggg gtactccgcg 540
gtgcacacaa cctcgactgc gtgttaagag agggtgccac gctattccct gtggttatta 600
cgacagtgga agacgccatg acacccaaag cattgaacag caaaatgttt gctgtcattg 660
aaggcgtgcg agaagacctc caacctcctt ctcaaagagg atccttcata cgaactctct 720
ctggacacag agtctatgga tatgctccag atggggtact tccactggag actgggagag 780
actacaccgt tgtcccaata gatgatgtct gggacgacag cattatgctg tcc 833
<210> 14
<211> 833
<212> DNA
<213>IBDV SD-F3 strains
<400> 14
cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60
ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120
agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact gactttcgtg 180
aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcggga gcattcggct 240
tcaaagacat aatccgggcc ataaggagga tagctgtgcc ggtggtctcc acattgttcc 300
cacctgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360
aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420
gccgcataag gcagctgact ctcgccgccg acaaggggta cgaggtagtc gcgaatctat 480
tccaggtgcc ccagaatccc gtagtcgacg ggattcttgc ttcacctggg gtactccgcg 540
gtgcacacaa cctcgactgc gtgttaagag agggtgccac gctattccct gtggttatta 600
cgacagtgga agacgccatg acacccaaag cattgaacag caaaatgttt gctgtcattg 660
aaggcgtgcg agaagacctc caacctccat ctcaaagagg atccttcata cgaactctct 720
ctggacgcag agtctatgga catgctccag atggggtact tccactggag actgggagag 780
actacaccgt tgtcccaata gatgatgtct gggacgacag cattatgctg tcc 833
<210> 15
<211> 833
<212> DNA
<213>IBDV GC-7 strains
<400> 15
cggtcgctgg ggtgagcaac ttcgagctga tcccaaatcc tgaactagca aagaacctgg 60
ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg atactgagtg 120
agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact gactttcgtg 180
aatacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga gcattcggct 240
tcaaagacat aatccgggcc ataaggagga tagctgtgcc ggtggtctcc acattgttcc 300
cacctgccgc tcccctagcc catgcaattg gggaaggtgt agactacctg ctgggcgatg 360
aggcacaggc tgcttcagga actgctcgag ccgcgtcagg aaaagcaaga gctgcctcag 420
gccgcataag gcagctgact ctcgccgccg acaaggggta cgaggtagtc gcgaatctat 480
tccaggtgcc ccagaatccc gtagtcgacg ggattcttgc ttcacctggg gtactccgcg 540
gtgcacacaa cctcgactgc gtgttaagag agggtgccac gctattccct gtggttatta 600
cgacagtgga agacgccatg acacccaaag cattgaacag caaaatgttt gctgtcattg 660
aaggcgtgcg agaagacctc caacctcctt ctcaaagagg atccttcata cgaactctct 720
ctggacacag agtctatgga tatgctccag gtggggtact tccactggag actgggagag 780
actacaccgt tgtcccaata gatgatgtct gggacgacag cattatgctg tcc 833
Claims (4)
1. differentiate the kit of chicken infectivity bursa of Fabricius virus highly-wetting liquid based on RT-PCR and RFLP technologies, it is characterised in that
Contain the primer pair and SpeI, SacI and StuI restriction enzyme for expanding chicken infectivity bursa of Fabricius virus VP 2 gene
Enzyme, the sequence of the described primer pair for expanding chicken infectivity bursa of Fabricius virus VP 2 gene are as follows：
PAU：5’ATCTTGGGTATGTGAGGCTG 3’
PAD：5’TATGGCCCGGATTATGTCTT 3’.
2. kit as claimed in claim 1, it is characterised in that also comprising rTaq enzymes, 10 × PCR buffer solutions, dNTP, RNase
Inhibitor, reverse transcriptase, 5 × RT buffer solutions and enzyme cutting buffering liquid.
3. kit as claimed in claim 1, it is characterised in that described kit is used to differentiate Bursal Disease
Carried out in accordance with the following methods during viral highly-wetting liquid：
(1) RNA extraction and reverse transcription is carried out to chicken infectivity bursa of Fabricius virus to be detected, obtains viral cDNA；
(2) PCR is expanded；
The cDNA obtained using step (1) is template, using PAU, PAD as upstream and downstream primer, the purpose base in PCR amplification VP2 sequences
Because fragment, reaction system are as follows：μ L of cDNA 4, μ L of rTaq enzymes 0.5, the μ L of 10 × PCR buffer solutions 2.5, dNTP 3 μ L, primer PAU
1 μ L, μ L of primer PAD 1, μ L of deionized water 13, the μ L of cumulative volume 25；
Wink is from being put into PCR instrument, amplification program is as follows after sample blending：95℃5min；94 DEG C of 30sec, 57.1 DEG C
45sec, 72 DEG C of 50sec, 30 circulations；72℃10min；4 DEG C of preservations；
(3) recovery and purifying of viral objective gene sequence PCR primer
The PCR primer of step (2) is subjected to purifying recovery, the DNA being collected into is stored in -20 DEG C；
(4) RFLP identifies IBDV strains
The PCR purified products obtained respectively to step (3) with tri- kinds of restriction enzymes of SpeI, SacI and StuI carry out digestion mirror
It is fixed；Digestion system is as follows：10U/uL SpeI or StuI or μ L of SacI 1, μ L of enzyme cutting buffering liquid 2,20~40ng/ μ L PCR is pure
Change μ L of product 7, the μ L of deionized water 10, the μ L of cumulative volume 20；
Mixing is put into 37 DEG C of water-baths after water-bath 2h, is mixed with 10 × Loading of 2uL Buffer, is coagulated with 0.8% agarose
Glue observes digestion result through 110V electrophoresis 20min；
(5) result judgement
If being cut out 531bp and 302bp two sections of fragments by SpeI, 242bp and 591bp two sections of fragments are cut out by StuI, without
Highly virulent strain can be then determined as by SacI digestions；
If only being cut out 218bp and 615bp two sections of fragments by SacI, and can not be then determined as by SpeI and StuI digestions
Low virulent strain.
4. the kit described in claim any one of 1-3 is preparing discriminating chicken infectivity bursa of Fabricius virus highly-wetting liquid reagent
In purposes.
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|---|---|---|---|
| CN201710703618.8A CN107475445B (en) | 2017-08-16 | 2017-08-16 | Kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies and application thereof |
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| CN201710703618.8A CN107475445B (en) | 2017-08-16 | 2017-08-16 | Kit for identifying virulent and attenuated strains of chicken infectious bursal disease virus based on RT-PCR and RFLP technologies and application thereof |
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Citations (5)
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| US6114112A (en) * | 1998-05-21 | 2000-09-05 | The Ohio State University | Method of making immunogenic compositions for infectious bursal disease virus |
| CN1265326A (en) * | 1999-12-23 | 2000-09-06 | 中国兽药监察所 | Triple live chicken vaccine and its production process |
| CN1522760A (en) * | 2003-09-04 | 2004-08-25 | 长江大学 | Bivalent gene engineering vaccine of fowl infectious bursal disease and pasteurellosis |
| CN101935637A (en) * | 2010-06-29 | 2011-01-05 | 中国农业科学院哈尔滨兽医研究所 | Recombinant low-virulent vaccine strain of chicken infectious bursal disease viruses (IBDV) and application thereof |
| CN103374631A (en) * | 2012-04-17 | 2013-10-30 | 中国农业大学 | RT-PCR (reverse transcription-polymerase chain reaction) method for identifying epidemic strains and vaccine strains of newcastle disease virus (NDV) |
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2017
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| US6114112A (en) * | 1998-05-21 | 2000-09-05 | The Ohio State University | Method of making immunogenic compositions for infectious bursal disease virus |
| CN1265326A (en) * | 1999-12-23 | 2000-09-06 | 中国兽药监察所 | Triple live chicken vaccine and its production process |
| CN1522760A (en) * | 2003-09-04 | 2004-08-25 | 长江大学 | Bivalent gene engineering vaccine of fowl infectious bursal disease and pasteurellosis |
| CN101935637A (en) * | 2010-06-29 | 2011-01-05 | 中国农业科学院哈尔滨兽医研究所 | Recombinant low-virulent vaccine strain of chicken infectious bursal disease viruses (IBDV) and application thereof |
| CN103374631A (en) * | 2012-04-17 | 2013-10-30 | 中国农业大学 | RT-PCR (reverse transcription-polymerase chain reaction) method for identifying epidemic strains and vaccine strains of newcastle disease virus (NDV) |
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| MARTÍN HERNÁNDEZ: "Novel Multiplex RT-PCR_RFLP Diagnostic Test to Differentiate Low- from HighPathogenic Strains and to Detect Reassortant Infectious Bursal Disease Virus", 《AVIAN DISEASES》 * |
| 杨蒙: "鸡传染性法氏囊病病毒强弱毒株的鉴别及VP2基因在大肠杆菌中的表达", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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