CN108660118A - One kind leading to the arthritic novel duck reovirus of duck and its application - Google Patents
One kind leading to the arthritic novel duck reovirus of duck and its application Download PDFInfo
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Abstract
The invention discloses one plant of duck reovirus, which is preserved in China typical culture collection center on April 26th, 2018, and deposit number is:CCTCC NO:V201818.Duck reovirus provided by the invention has good immunogenicity to currently a popular by the duck reovirus of chief complaint of gambrel swelling.Good with inactivated vaccine safety prepared by the duck reovirus strain of the present invention, protective rate can provide protection completely up to 100% to the variant newly detached.
Description
Technical field
The present invention relates to the separation of Avianreovirus strain and application fields, and in particular to one kind causes duck arthritic
Novel duck reovirus and its application.
Background technology
Avianreovirus (Avianreovirus, abbreviation ARV) belongs to Hu Changgubing sections (Reovirdae) and is just exhaling intestines lonely
Tobamovirus (Orthoreovirus) can cause birds that a variety of diseases occur, and clinical manifestation is because of virus stain, virulence or sense
Contaminate the different and variant of host.Currently, the main means of control fowl group's ARV morbidities are using attenuated live vaccine or the full disease of inactivation
Malicious vaccine controls typical Avianreovirus infection by immunity inoculation.But since Avianreovirus is RNA virus, have
Multiple segmentations exist in antigenic structure, pathogenic, cell culture characteristic and host specificity etc. between different strains certain
Difference.Variation is easy to happen in genetic evolution, cause vaccine prepared by traditional strain cannot well prevention and control it is popular now
Avianreovirus infection.
From 2016, the outburst on a large scale of the ground such as China Shandong, Hebei, Henan and Jiangsu duck is with gambrel enlargement for main disease
The communicable disease of shape, the disease mainly cause each age level duck gambrel swelling, gambrel often to contain a small amount of yellow or blood
Sample exudate;Several cases have cheesy exudate in articular cavity, suffer from duck and different degrees of limping occur.The disease mainly results in
The decline that kind duck is laid eggs with meat duck weight, feedstuff-meat ratio increase, and meat duck delivers qualification rate significant decrease for sale, to meat duck and kind duck cultivation
Industry causes serious financial consequences.Using commercially available commercialization duck reovirus vaccine can not the effective prevention and control diseases stream
Row, thus deduces that the disease may be caused by novel reovirus.Therefore, start with from cause of disease, further study molecule and cause
Anttdisease Mechanism and virus variation mechanism, it is the key that the prevention and control disease to develop corresponding vaccine.
Invention content
For the above-mentioned prior art, the object of the present invention is to provide one kind leading to the arthritic novel duck reovirus of duck
And its application.
Specifically, the present invention relates to following technical schemes:
The first aspect of the present invention, provides one plant of duck reovirus, which is preserved on April 26th, 2018
China typical culture collection center (abbreviation CCTCC), address are:Wuhan, China, Wuhan University, deposit number are:CCTCC
NO:V201818.
The second aspect of the present invention provides above-mentioned duck reovirus and is preparing answering in preventing the vaccine that duck exhales intestines orphan sick
With;It is using the swelling of duck gambrel as cardinal symptom that the duck, which exhales intestines orphan's disease,.
Preferably, the vaccine is inactivated vaccine or attenuated live vaccine;It is furthermore preferred that the vaccine is inactivated vaccine.
The third aspect of the present invention provides above-mentioned duck reovirus in preparing treatment duck and exhaling the lonely sick Yolk antibody of intestines
Application;It is using the swelling of duck gambrel as cardinal symptom that the duck, which exhales intestines orphan's disease,.
Beneficial effects of the present invention:
Novel duck reovirus provided by the invention is exhaled by the duck of chief complaint of gambrel swelling currently a popular
The lonely virus of intestines has good immunogenicity.Good, the protection with inactivated vaccine safety prepared by the novel duck reovirus strain
Rate can provide protection completely up to 100% to the duck reovirus variant newly detached.
Description of the drawings
Fig. 1:Cytopathy of the virus isolated strain of the present invention on LMH cells;Wherein, A:Under normal growth state
LMH cells;B:The CPE that N-DAV-SD16 occurs on LMH cells.
Fig. 2:PCR qualification results;Wherein, M Marker;Swimming lane 1 is sample to be tested;Swimming lane 2 is blank control.
Fig. 3:The sequence analysis result of L1 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Fig. 4:The sequence analysis result of L2 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Fig. 5:The sequence analysis result of L3 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Fig. 6:The sequence analysis result of M1 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Fig. 7:The sequence analysis result of M2 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Fig. 8:The sequence analysis result of M3 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Fig. 9:The sequence analysis result of S1 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Figure 10:The sequence analysis result of S2 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Figure 11:The sequence analysis result of S3 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Figure 12:The sequence analysis result of S4 genes in the virus isolated strain N-DAV-SD16 viral genomes of the present invention.
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology part is introduced, since Avianreovirus is RNA virus, there are multiple segmentations, different poison
It is had a certain difference in antigenic structure, pathogenic, cell culture characteristic and host specificity etc. between strain.Heredity into
It is easy to happen variation when change, causes vaccine prepared by traditional strain cannot the current popular Avianreovirus sense of prevention and control well
Dye.From 2016, the outburst on a large scale of the ground such as China Shandong, Hebei, Henan and Jiangsu duck is using gambrel enlargement as cardinal symptom
Communicable disease.The instep of symptom caused by the symptom of communicable disease duck reovirus different from the past, sick duck closes
There is enlargement in section, is prevented using commercially available commercialization duck reovirus vaccine, can not be realized very to the communicable disease
Good prevention and control.Thus deduce, which may be caused by novel reovirus.
Present inventor isolates one plant of duck reovirus N-DAV-SD16 from the tendon tissue of morbidity duck swelling,
Birds reovirus gene by segmented 10 genetic fragments (including:L1-L3, M1-M3, S1-S4) composition, the present invention couple
The duck reovirus N-DAV-SD16 of above-mentioned new separation has carried out genome sequencing, and respectively by 10 genetic fragments and now
The Avianreovirus having been reported that has carried out sequence alignment and homology analysis, as a result, it has been found that, new isolated strain N-DAV-SD16
10 genetic fragments in L1, L2, L3, M1, M2, S3, S4 be respectively positioned in a relatively independent branch, and at present on
Biography reovirus-originated duck homology in Genbank is relatively low, illustrates that the strain N-DAV-SD16 newly detached is different from others
Avianreovirus, it can be assumed that duck reovirus found in the present invention with it is existing it has been reported that duck reovirus
In the presence of compared with Big mutation rate, being 1 independent kind of Orthoreovirus.By further comparing discovery, which exhales the lonely disease of intestines
Part has been recombinated in malicious N-DAV-SD16 genomes leads to the S1 gene pieces of broiler chicken source reovirus of broiler chicken gambrel swelling
Section, therefore conduct a research and be of great significance to the novel duck reovirus.
Compared with existing Avianreovirus, 10 in duck reovirus N-DAV-SD16 full-length genomes of the invention
Genetic recombination and mutation has occurred in a genetic fragment, enhances the immunogene of the duck reovirus N-DAV-SD16 of the present invention
Property, the effective prevention for exhaling currently a popular duck the lonely disease of intestines can be realized by being made into vaccine.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.If the experiment actual conditions being not specified in embodiment, usually according to
Normal condition, or the condition recommended according to Reagent Company;Reagent as used in the following examples, consumptive material etc., such as without special
Illustrate, can be obtained by commercial sources.
Wherein, it is century Co., Ltd, the purchase of plain agar sugar gel reclaims kit that RNA extracts kits, which are purchased from Beijing health,
In Beijing Quan Shi King Companies, reverse transcription reagent box is purchased from precious biological (Dalian) Co., Ltd, and 2 × Es Taq MasterMix are purchased from
Beijing health is century Co., Ltd, and DL2000Marker is purchased from precious biological (Dalian) Co., Ltd, fetal calf serum, DMEM culture mediums
BI companies of Israel are purchased from, PBS is purchased from Beijing Suo Laibao companies.
Embodiment 1:The separation and identification of duck reovirus strains
1. virus purification:
(1) it clinically is accredited as positive arthritic duck through PCR reactions, the tendon tissue that dissect acquires its swelling is set
In 15mL centrifuge tubes, be added 5 times of volumes serum-free DMEM culture mediums, multigelation 3 times after homogenate, every time thaw after
Freeze thawing next time is being carried out after shaking 1-2min on oscillator;After freeze thawing by the 15mL centrifuge tubes equipped with sample in centrifuge
4000rpm centrifuges 5min;Take supernatant, it is spare after 0.22 μm of filtering with microporous membrane;
(2) according to the multiplication characteristic for exhaling the lonely disease of intestines, chicken gizzard cancer cell (LMH) is selected to carry out virus purification.According to conventional thin
Born of the same parents' cultural method, waits for 25cm2When cell confluent monolayers in cell bottle, suction, which is abandoned in bottle culture medium and swung with PBS, washes cell 2 times,
The sample freeze thawing supernatant that 0.5 milliliter of step (1) has filtered is added, cell is placed in 37 DEG C, 5%CO2Feel in the incubator of concentration
Make 30min, sense is added the DMEM culture mediums containing 2% fetal calf serum after making, observes and records the time for cytopathy occur.
If the acellular lesion of 1st generation occurs, then the culture harvested with 1st generation after freeze thawing continues to detach according to step (1) method, until
It obtains and stablizes strain;The 5th generation still acellular lesion is such as reached, then is considered as virus purification feminine gender.Finally obtain one plant of stable poison
Strain, occurs more apparent cytopathy for 3 days after being inoculated with LMH cells, that is, shows as cell rounding, fusion, the group of presentation
Block falls off lesion (Fig. 1), normal with batch blank control cell, which is named as N-DAV-SD16.
2. viruses indentification:
(1) PCR is identified
1) RNA is extracted:
By the virus liquid of strain N-DAV-SD16 according to the requirement of RNA extracts kit specifications, viral RNA is extracted, be placed in-
20 DEG C save backup;
2) reverse transcription obtains cDNA:
Reverse transcription reagent box used uses the article No. from precious biological (Dalian) Co., Ltd for RR036A's
PrimeScriptTMRT Master Mix sequentially add 5 × PrimeScript RT Master in 200 μ LPCR reaction tubes
The μ L of Mix × 2, the middle sample to be tested Total μ L of RNA~2 extracted of step 1), use RNase Free dH2O is supplemented to 10 μ l bodies
System.It is placed in PCR instrument and is reacted, reaction condition is 37 DEG C of 15min;4 DEG C of preservations after 85 DEG C of 5s.
3) PCR amplification:
It is expanded using 20 μ L systems:The μ L of template cDNA × 2, upstream and downstream primer is each × 1 μ L, 2 × Es Taq
The μ L of MasterMix × 10, using ddH2O is supplemented to 20 μ L systems.Mixing is simultaneously reacted in wink from PCR instrument is placed on, and reacts item
Part is 95 DEG C, 5min, and 95 DEG C later, 45s, 56 DEG C, 30s, 72 DEG C, 30s carries out 30 cycles, 72 DEG C, is protected for 4 DEG C after 10min
It deposits spare.
Sense primer:5′-TGA GAC GCC TGA CTA CGA TT-3′(SEQ ID NO.1);
Downstream primer:5′-ATG CTT GGA GTG AGA CGA CT-3′(SEQ ID NO.2).
4) PCR qualification results:
Product after carrying out PCR amplification with primer occurs big with desired value 513bp in 1% Ago-Gel after electrophoresis
The small specific band (Fig. 2) being consistent.Show that there are novel reoviruses in the cell culture of each separation strains.In addition to dividing
From the conventional duck source viral diagnosis that strain carries out, and other virus pollutions are not detected.
(2) sequencing:
Whole genome sequence measurement carried out to strain N-DAV-SD16 using two generation sequencing technologies, in whole genome sequence
The sequence of this 10 genetic fragments of L1, L2, L3, M1, M2, M3, S1, S2, S3, S4 is respectively such as SEQ ID NO.3-SEQ ID
Shown in NO.12.Then sequence analysis, strain N-DAV- are carried out with the Avianreovirus sequence announced in the prior art
The sequence analysis result of 10 genetic fragments of SD16 is respectively as shown in Fig. 3-Figure 12.
As seen from the figure, 10 genetic fragment multidigits of strain N-DAV-SD16 are in a relatively independent branch,
Illustrate that the strain N-DAV-SD16 newly detached is different from other Avianreovirus, is that 1 of Orthoreovirus is independent
Kind.
Isolated strain N-DAV-SD16 is accredited as duck reovirus.And biological deposits are carried out to the strain,
Preservation information is as follows:
Strain name:Duck reovirus
Classification And Nomenclature:N-DAV-SD16 plants of duck reovirus
Preservation mechanism:China typical culture collection center
Preservation mechanism is referred to as:CCTCC
Address:Wuhan, China Wuhan University
Preservation date:2018.4.26
Collection is registered on the books number:CCTCC NO:V201818.
Embodiment 2:TCID50Titration:
1. test method:
The 5th generation cell cultures of strain N-DAV-SD16 (the DMEM culture mediums for containing 2% fetal calf serum) are done into doubling dilution
(10-1-10-6).After the LMH cells of 96 porocyte culture plates are paved with each hole, each dilution ladders of 25 μ L are sequentially added in order
The virus liquid of degree, most latter two hole are added DMEM culture mediums of the 25 μ L containing 2% fetal calf serum and do blank control, in same cell version
8 repetitions are done, are placed in 37 DEG C, contain 5%CO2Incubator in after sense makees 30min, 100 μ L are added per hole containing 2% fetal calf serum
DMEM culture mediums continue culture observation, observe and record the case where cytopathy occur daily, until the 5th day sentence read result.With Reed-
Muench methods calculate separation strains TCID50Potency.
2. test result:
After measured, the TCID of strain N-DAV-SD1650Titration result is:10-4/0.2mL。
Embodiment 3:Animal Orthogonal Rotational Regressive Tests
Cause of disease of learning from else's experience and antibody test do not carry the 1 age in days duckling 40 of health of novel reovirus and antiviral antibody
Only, it is divided into 2 groups, every group 20 at random.Wherein the 1st group is experimental group, and experimental group uses the 5th generation cells of strain N-DAV-SD16
Culture is through footpad inoculation, and only, the 2nd group is control group to 0.5mL/, and every footpad inoculation DMEM culture medium as a contrast, raise by isolation
It supports.The state of mind of each group animal is observed after inoculation daily and gambrel swelling situation and is kept a record.
Experimental group starts gambrel swelling lesion occur for 7 days after virus inoculation, the 9th day after inoculation, 20 experimental groups
There is gambrel swelling lesion, identical as natural infection pathological conditions, control group is in a good state of health.Take experimental group lesion instep
Joint operates progress cause of disease according to above-mentioned pathogen separation and detaches again, being capable of isolated novel reovirus.
Embodiment 4:The preparation of inactivated vaccine
(1) viral proliferation and harvest:
The novel duck reovirus N-DAV-SD16 being separated and identified is inoculated into well-grown LMH cell lines,
The virus is largely proliferated, enough virus liquids are obtained.The sufficient cells virus liquid of acquisition is placed in -20 DEG C of freezings, is passed through
Twice after freeze thawing, cytopathy venom is collected.
(2) viral purifying:
With detection methods such as established PCR, RT-PCR, detect in the virus liquid of acquisition whether contain other common virus.
Detection project include avian influenza virus (AIV), newcastle disease virus (NDV), Duck parvovirus (DPV), goose parvovirus (GPV),
Infectious bronchitis virus (IBV), infectious bursa of Fabricius virus (IBDV) etc., and kind of a poison is purified.
(3) inactivation of virus liquid:
The qualified virus liquid of bacterium inspection is inactivated with formaldehyde, and best inactivation condition is that the first of final concentration 0.2% is added
Aldehyde, 37 DEG C of stirring inactivation 16h.
(4) preparation of vaccine:
1. the preparation of oil phase:Take No. 10 medicinal white oils, aluminum stearate (Aluminum tristearate), Span-80
(Span-80) 94 are pressed:2:6 mixing, after stirring and evenly mixing, autoclave sterilization.
2. the preparation of water phase:Tween-80 (Tween-80) is added in the antigen liquid of inactivation, mixing is vibrated, makes Tween-80
It thoroughly dissolves, the final concentration of 2-4% of Tween-80 in water phase.
3. emulsifying:Oil phase and water phase press 2:2 parts of oil phases are added tissue refiner in super-clean bench, delayed by 1 ratio mixing
It slowly by 1 part of water phase, is during which stirred continuously, 6000rpm mixing 10min after water phase is all added, then 8000r/min emulsifications
20min, packing is spare, that is, inactivated vaccine is prepared.
Embodiment 5:The quality inspection of inactivated vaccine
Include by inactivated vaccine prepared by embodiment 4:Dosage form, centrifugal stability, viscosity, sterile and storage life matter
Amount is examined, specific method reference《Republic of China Veterinary Pharmacopoeia》(2015 editions).
As a result it is:The dosage form of inactivated vaccine prepared by the present invention is water-in-oil type (W/O);Centrifugal stability, viscosity and nothing
Bacterium, which checks, to be met《Republic of China Veterinary Pharmacopoeia》The regulation of (2015 editions).
Embodiment 6:The safety examination of inactivated vaccine
1 age in days duckling 20 is taken, is divided into 2 groups, every group 10 at random.Wherein the 1st group is experimental group, leg muscle injection
Inactivated vaccine prepared by embodiment 4, only, the 2nd group is control group to 0.5mL/, and leg muscle is injected equivalent sterilizing white-oil adjuvant, connect
The state of mind of each group animal is observed daily after kind and whether injection site the local inflammation reactions such as red and swollen heat pain occurs, is held
Continuous observation dissects experimental animal after 2 weeks, 2 weeks, observes injection site vaccine absorbing state.
As a result:It is depressed that of short duration spirit occurs in experimental group, but restores quickly, continuous observation two weeks, test group and control group
Growth and development is normal, and the state of mind is good, and it is good that dissect test group finds that injection site vaccine absorbs, and no redness, tissue are bad
It waits indefinitely inflammatory reaction.As a result prove that trial-production vaccine safety is harmless, on growth of animal without influence.
Embodiment 7:The protectiveness of inactivated vaccine is examined
1 age in days duckling 60 is taken, is divided into 2 groups, every group 30 at random.Wherein the 1st group is immune group, leg muscle injection
Inactivated vaccine prepared by embodiment 4, only, the 2nd group is control group to 0.5mL/, and leg muscle injects equivalent sterilizing white-oil adjuvant,
10 days after immune, 0.2mL novel duck reovirus liquid injected to two groups of duckling legs, and be grouped isolated rearing, observe two groups
The mental status and death condition of duckling.
As a result:It is depressed that of short duration spirit occurs in immune group, but does not show the clinical symptoms of duck reovirus disease, does not go out
Existing death;There are apparent clinical symptoms (gambrel swelling) after attacking poison in the duckling of control group, and dead number of elements is 8, as a result
It has been shown that, the immune protective rate of the inactivated vaccine can reach 100%.
Embodiment 8:The immune duration of inactivated vaccine measures
1 age in days duckling 20 is taken, is divided into 2 groups, every group 10 at random.Wherein the 1st group is immune group, every leg muscle
Inactivated vaccine prepared by embodiment 4 is injected, 0.5mL/ is only;2nd group is control group, and leg muscle injects equivalent sterilizing white oil assistant
Agent;Serum is acquired after immune every three days, antibody is detected using indirect ELISA.
As a result, it has been found that the antibody level of immune group on day 4 when start significantly to increase, peak within the 10th day, this
It is slightly decreased in 30 days afterwards but still maintains higher level;And the antibody level of control group is feminine gender in entire experiment.Explanation
The inactivated vaccine of the present invention can be rapidly achieved the antibody level of high concentration in a short time, and immune duration is long, Neng Gouwei
Duckling provides quick, long-term immunoprotection.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>One kind leading to the arthritic novel duck reovirus of duck and its application
<130> 2018
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
tgagacgcct gactacgatt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
atgcttggag tgagacgact 20
<210> 3
<211> 3928
<212> DNA
<213>L1 genetic fragments
<400> 3
ctttttctcc gatctccgaa atgagttcgc gcaaagtggc tagacgtcgt cataaggatg 60
ctactgaatt taaatacact aagaacgtta acaagtctaa gccatcctct actgacgtta 120
aagaacctgc ggatagtgcc acagataaga aagtcactgt tccatcacca gacaatccag 180
ccgcttctac tccctcttcc actgatggag cttctcaaac atccgttgct aagcagacga 240
atgataatga cgcctcagtt aaggaatcag ctcccaagcc taccgtatct agcgatggga 300
aagacggaat gcatggtgct gtgaagttgc aagacgctaa ggccactgca gctgtggata 360
gtaataagga tagagatgtg gtatttggtg gcgcaggctc tggtgacaaa aatgctatta 420
cgaagactgg ttccgttgac aatgatgggg gcgttaaagt cgttccagcc aaggatgcta 480
cgatatcttc ggccaaagcc atgatggaac agaagcagtt agttgctggt cttccgaagc 540
aaccgaagtc tgctaatcat ttgtgcaccg tttgtatggc acaattcgcg tctgctgacg 600
ccctaactat ccatcagact acgcactcca ttggctccaa tgctgctctg acgagcttct 660
cgatttctac tgctgttgaa gaattcattc aatcatgggc tgctgctacg tccactgcca 720
ataccaagac ggccttaact gtgtctgacg tggactcgct gatgatgact gaaggaatac 780
gtctcataac ttgggattct gggttatgta catcttttga gcttgtcccg attgtccatt 840
caaacactgt tcaagatgta atttcgtact catggttcac atcaagttat aatatcacaa 900
ctcccttccc acaggcgcct gtcgtgcgaa ttgttttacg tactaattgg gctgccaaat 960
tggactctcc ctcgtcgtcg cgtgaatgtg atcttcgcct cgccccacct acagagagta 1020
atgctcgatc attctcaatg ctgctcaata ctggtgcgac tccagaaggt actttcaacc 1080
ccaacaccct tcgtatgaat gtgctgcaga tgtgtcttca gtatgttctg gctaacctac 1140
acttgaatcg tagcactcag ttcaccatgg atttgactgc cgcagctccc aatctgtcgg 1200
cgtctcaact ccgtatcgtt ccagatgata aagatggtaa atggttccct gtcatgtatc 1260
catctcgcgt gaacatccca ctgttcaata agacagctga ttttgttaat cagtgcattc 1320
gtgacagaat tggccgatat gaccgcgccc agactttcgc tggcgcaccc tctgaatggg 1380
ctgacatgtg ggaaacagcg gacgcgttaa ctctctccgt ccgtgaaatg tggatgtcac 1440
gtatttccca aatgaatatc tctcctgctg atatcgctga cgctatctct cgatgttctc 1500
agtccctgct cactgttgcc gcgccaacag ctccttctgt agctcgtctg ttaccttggc 1560
gggttagttc cgatgagagg cagctcctcc aattgttgat gtacttgaac gttgggacta 1620
gtgccgacta cgtccaacct attctgtctg cgttcgctcg aactctatct cgcgtgtcac 1680
cattgcgtat taatcccact ttaatcgcta atgccatgtc gacaattgtc gagagcacta 1740
ctaataccca gagtcctgcg gcagctattt tgtcgaagct taaacctgtt gcctcggact 1800
tctccgattt taggttggcg tgtgccgctt ggttgtataa tggttgcgtc cagacatact 1860
tgtctgagga ttcatatcca agcagtggtg gatctgttac tagcattgat acattggttg 1920
atatgtttgt gtgcttgttg gctttgcctc ttgttactga tcctaatgct ccttgccaag 1980
cctttatggt tgtcgctaat gccatggttg gttacgagaa tctaccgatg gacgacccta 2040
attttactca gcaaagattg gctgcagcgt tcaacaatcc cacgacctgg cctcaatgtt 2100
tcctccaccc tcaaaacatc gatcgacgcc aatgtccgat tctctcatgg tgggctcagc 2160
agattcatcg taattggccc acaccgtctc aaattactta tggggcgcct gacatcattg 2220
ggtccgctaa cttgtttact cctcctgacg tgctgttgct tccattacag cataggccca 2280
tccgtattac taatcccacc ctgaacttcg ataatgagtt gacgacttgg cgtaacaccg 2340
tggttgatct ggttctgcgc atcatcgaca gtggtcggta ccagcccaat tggaatcagt 2400
ccatccgtgc ctctatgcgg aatgcgatga caaatttcag aattattaag tcctatactc 2460
ctgcttacat agcagaactg ctacctgtgg aactggcagc tatcgctcca actctaccct 2520
tccagccttt tcaggtgccg tttgcccgct tagatcgcga cgctatcgtc actcacgtca 2580
atgtatctag acaagctccc aacaatcttg ctcaacctgc attgaacatg tccatgacgt 2640
accagcgcac aggggttcca atctctctta gtgcccgtcc cttggcagtc gctcttctgt 2700
caggccagta ccctactgat ccccctcttc agactaatgt ttggtacgta aacactctca 2760
cgcctctata ttccaatgat ggtctcttta ataacgtcca gcacgctatg gttgcttctg 2820
aagcttacgc taccttgatc accatgctgg ctcagtgcac tgacatgcag taccctgtgg 2880
atcggccatt gaattggctt cgtcagatta atttggctgc taatgaggcg acgattttcg 2940
gtcgatcaat taattcactt ttccagactg ctttcgacct ttcaccttcc actgtattgc 3000
ttcaaccgtt tttggaatct gatccacgtg cgacacagct agccatttct tacgttcgtt 3060
ataatggtga cagtgaaacc ttcgtgccaa cagtgcgtcc atctatgatt tcagaagcga 3120
cattgctcgt tgagcgtact ctctcgcacg aatacaacct cttcggttta tgccgtggtg 3180
acatcatcct gggacagcac atgactccaa ctgcgttcaa tcctttggct ccgcctcctt 3240
ccgtcgtttt taacaggggt gatactgatg tctatgagtt tggctctcgt agcttcgcca 3300
acttcggcat gaatggggag gagatcttgg tcatggatgc gaacggcgtg cgtcgtccat 3360
tactcggccg gtgggttatg ccactgcagc ttttgatggt taatattggc gtatttccta 3420
agctgttgct ggatcgtatc ctgaaggggc gcttgtatat ccgacttgaa gttggcgcgt 3480
atccatatac ggtgcagtat taccagggac gtgagtttac ggatggcttc actctgcttg 3540
agcaatggat gtctaaggtg tcgcccatgg gtatccctcc cgtccctttc ctcatgccgc 3600
agtccgaagg acacaacatc acttcaggca tggttactca ttacatctgg tccactgaat 3660
ataatgatgg gtccctcttc gccacgaaca ccgacctacc agttactgtg ttcggccctg 3720
accgcaccat cccaatcgag cgttatcggg cactcgtgga tccaggtgct cttcccgcta 3780
ctaaccaact gccgcacacc attgatcttt actgctcact gagacggtac tacttggaga 3840
cacctcccat cactgcgacc gtcaccactt atggcgatgg actccccgcg ctgaaccatt 3900
agagcggcga ggctagacgc gagctgat 3928
<210> 4
<211> 3830
<212> DNA
<213>L2 genetic fragments
<400> 4
gctttttcct caccatgcat gtcaacgggt ttgatgatgc tactctctcc tacgcacagt 60
ccatatcggg ggttatccca atgacaaata agttatttga gcaagcatct acctcaatac 120
gtgccctacc acgctcacat gtttatgcta tattagataa tgttaatttt tcagtttcat 180
gtgtcatccc gaatcgtatt ttccatcatc ctgatcactc tgagtacttt tacattgatg 240
cggttaacag agttagacgg aaacaagtta tcgatcctga tgatgtattt gtgccaaact 300
gcaatttgca gggtcttatt actccaatgg agagattacc aaattatggt caattgtctg 360
agactatttc gtcgaacgct cgggatggct tgccatctgc acgcgtagca gctacttttt 420
ataacacttc ggtgtcacag gctcgccaag ttaaagctcc acttgaatcc tttttgttgc 480
ctttgctatt atctgagact tgctcattat cagacgatcc ttgcggactt gacaccgcag 540
cttctcctcc gatccatacc aatctggcgt tatgggtgtt acgcgaaatt agtcgaacta 600
tttgtggatc ttcgaatgac cgttcacctt ggttactgct cgattcaggg gttgcgtggt 660
tcatgtctcc gttaatgtca tcggccattc cacctcttat ggctgactta acgaatctag 720
cgatctacaa acaaatttgt tctgtgtctg atgagcttca ttcccttgca gttcaagtgg 780
ttttgcaggc cgcggcgtca caatcgtatg gacactacat attgcagacg aagtcagtat 840
tcccccaaaa tacgttacat aacatgtttc gtacgctcac tgatggtatt gttccggtca 900
tagattggtt agaaccgcgt tcaaactatc gcttcatgct tcaaggtgcg cgtaaagtga 960
cttcagatga tgcgaatcag gctccggata acacggaagc cgcggagcaa ctcggtcgca 1020
agatggggtg cctcgatgtc gtacgctcct tacgtaagat gtcctcgtcc atcactgtcc 1080
attcacacga tgccatgacc ttcgtacgtg atgctatgtc gtgcactagt ggcatcttta 1140
tcacgcgtca acctactgag accgttttaa aagagtacac tcaagcgccg accattgaag 1200
tccctattcc tcaatcggac tggtcaccgc ctattggatc tctgcgatat ctctctgatg 1260
cctgttccct ccccgctgtg tatctggcta gggcttggcg cagagctgcc tctgctgtag 1320
tagataatcc gcacacttgg gaccctttat atcaggccat ccttcgctct caatatgtga 1380
cgtcacgcgg tgggtccggt gccgcgttaa gagatgcttt gaaggctgcg gaagttgagc 1440
ttcctcagta tcctggggtc agtgttaaag tggcgaccaa gatttatcaa gcggctcaga 1500
ctgctgacgt gcctttcgat aaattatctc gtgctgttct agctccattg tcgatgggct 1560
tacgtaacca agttcagcga cgtccaagaa ccatcatgcc catgaatgtg gttcaacagc 1620
agatttcagc ggctcatact ctctccgctg actacattaa ttatcacatg aacttgtcga 1680
cgacctcggg tagcgctgtg attgagaagg tggttccatt aggtatgtat gcatcctgtc 1740
ctcccgctca agcggttaat attgatatta aggcttgtga cgcgtccatc acgtaccagt 1800
attttctttc tgttatcgtc ggtgccattc atgagggtgc agcaggccgt cgtgtctcgt 1860
cttcattcat gggcgttcca ccaagcgtgc tgtccgttgt cgatgctagc ggagtgactt 1920
catccatgcc catctcaggt tttcaagtca tgtgtcaatg gttggctaaa ctttaccagc 1980
gaggttttga gtatcaagtg acggatacat tctcacctgg caatattttc acgcatcaca 2040
ctactacttt tccctctggt tcaacagcga cgtctacaga acatactgcc aataatagca 2100
cgatgatgga tggcttcctg cgcgcttgga ttccttcctc cggtgcgtcc gacgtactga 2160
agaagttctg caaatccatc tcaatacaac ggaattacgt ttgtcagggt gacgatggtc 2220
taatggtcgt tgatgggcta tcgacaggta aattatcagg cgagataatc gatgaatttg 2280
ttaaggaatt gagagcctat ggtaaatcat ttgggtggaa ctatgatata gagtttaccg 2340
gaaatgcgga gtatctaaag ttgtatttcc taaacggttg ccgtatacct aatgtttctc 2400
gacatccgat ctgtggcaaa gagcgcgctt caggggacaa gttagaaatg tggccgtcca 2460
ccattgacat cttcaatggc atatttgtga atggtgtgca tgatggtttg ccgtggcgca 2520
gatggttacg ttattgttgg gctcttgctc ttatgtattc tggaaagacc gtgcgtcatg 2580
atgattctga ggtgttgatc caatatccta tgtggtcttt tgtgtattgg ggtttgcctc 2640
ccgtgagcgc gtttgggtct gatccatgga tcttttctcc atatatgccc actggtgatc 2700
atggtttcta ttcaatgttg actttagtgc gccctctgat cactaacttg tccccatcgt 2760
cagacgcttc gggattattt ggtcaatgcg atcacaacgt tttgttcaat tctgagctag 2820
tttatcaggg ctattacatg gctcaatgcc cacggcaacc ttctcgctcg aaccgtagag 2880
atgatcccga ctctgtacag cgcttcgtca aggccttgga gtcttacctt tacatttccc 2940
ctgagctaaa atcgcgagtg cgccttggtc gtgaccgctg gcagaagttg gttgggtata 3000
cggaaaaatc tcccccgtcg cttgatgacg tggcgttcaa atggttccgt agtgcacagg 3060
aagctgatct cccaaccgct acagagattc aaagcatgga tctggccttg ctggcagcca 3120
gacgtaggac atatcagggc ttctccaagt tgttgaatac ctatttaagg gtaacctggg 3180
atttatctga tcctgttgaa cacgctgtag atccccgcgt acccttgtgt gccggtgtct 3240
ctccatcaaa tagcgagccg ttcctcaaat tgtactccgt aggtcctatg atgcaatcca 3300
cgcgtaaata ctttagcaat acgctattta tccatcggac tgtgtctggt cttgacgtcg 3360
atgtcgtcga tcgtgcgctc cttaggttgc gtgcccttaa tgcgcctgat gacgtggttg 3420
tagctcaact tttgatggta gggttgtctg aagccgaagc cgctacatta gcagcgaaaa 3480
tacggacgat ggatatcaat gccgtgcaat tggccagagt tgtcaactta tccatccctg 3540
actcatggat gaccatggat tttgatcgct tgatacgaga tatcgtgtct gtcactcctc 3600
tgaccgttcg atccctaacc accgatctac cctctggcgt accgtgggct cgcgcgatct 3660
tacagttctt aggtgcgggt gtcgccatga cggccgtcgg acccttgcgt cgtccctact 3720
tacactcagt tgccggaggc atgtcttcat tcattaagca gttccgccgg tggatgcgtg 3780
ccgaaacgag gtagcgtccg tgcccggcat ggctcgagga attactcatc 3830
<210> 5
<211> 3876
<212> DNA
<213>L3 genetic fragments
<400> 5
gcttttccac ccatggctca gattcgaggc cttcggttgt ctacgacgct ctcagctcca 60
cctccacgca agattataac atctcacact tatgatgagc tgatctccgc tctgaagtta 120
gcaaccaagc cttggcgccc tttgaagtcg cgaaataatg attctgtcac ggcagtgcag 180
ctcctttttc cccttaatgg ttatattgaa cccatgttca tgttggaaaa ggatatgacc 240
cttagtgatt ttgaggcctg gttgacgcct cttctatctg cactcgctga ccagttgctt 300
agacactacc ctatcgccgc ctatcacggg cgtttgatta atccgctgct atctaatgca 360
attgttgccg ctttcttgtc taacgtgcct tatgcgcatg cattggatca tctcttcctt 420
gttagaggaa acgtcgagga tattatggat gcggggatcg caattcagaa tcacttgtgg 480
ttcgatcgcg gtgcactagt gacccctgct ggacagaaat ttgttcagct gactggctat 540
aacttctcct ctaatgatcc gtgtctattt tctaagcaat tgcgttgtta tggcctcgtt 600
tactactttc tcgacatggc cgaatgtctg gcgtattgtt ggcgtcatct atccaattca 660
actccactga tacactttga ccgtccgtcc aatggagttc attgtttggt gccctctgaa 720
tccacgacgc ctatcgctgg ttcgttacca gtgtcagcac tcagctctat tttgttggaa 780
tcctgtcttc agcaatctac aattaatgcc cttactccca ctggttcgcc cgtcattaga 840
caaatagaag cattgttgcc tatatcatca ccgtttttcg aacgacggaa cactctggaa 900
tattctctct tcgcactgtc aaatgctctg gtaaatggtt atcagcttgt agacttgcgt 960
tccggccacc ctgattgcgc tactgttgct gccatcctag ctagattgat tgatttctct 1020
aaggatatca ccgttattca accgcgccct gctctctttg ctatcaacag cgacagtccg 1080
cttacgtata gtggagaaaa tgctaatttt atttcgcgct tgacttgtgc gtccggtaga 1140
cctattggtc cggtcgttgt cggtaaatcc gttgatcatg ccgttggttg gatgccccag 1200
tttgaccccg ccacgtccta caaccctgat ctctcgatgg actcacttgc tcgtgccacg 1260
acactgcctc tccgtgctaa gtattcgact ttctggtcag gcccagcatt gttttctttc 1320
gcttcatgta ataggcacaa tggtgtatat gacatacagt tcatggctca atttcctcct 1380
acttacttta gtgatgatga cgccttttct agatcacgat tctcttctta tcgtgcagtt 1440
agggaccggt cattgttgaa ggataccgct aatttgatgt acatctcgaa tttgtccagc 1500
tctcacgacc atcgtcttgt cccagattct aaaactatga tttatgtggg ttcctctggt 1560
actcatgtag ataatcaacc ttctatcatt aaacctctct tagctggaac tcttccgggt 1620
gtttttcgcc ccctttctat aaaacaggtt ggttgggagg tcactaatgg aacgatttgt 1680
gatgttgagc ttcctttagc cactggtacg ttcttcttcg tgtacagtga tgtggatcaa 1740
gtgcaatcag gtgattctga tttggacgcc tcctcgcgtc gcttttgctc ccaattggac 1800
atgctaatga agttgacgtt tactggtgga tcgcttgtcg ttaaatgcaa ttttccgact 1860
agtctagtgt ggcgtcacat cttctccact gtttctcctt atttctcggc tattcattta 1920
atgaagcctc tcgtgtcaaa taatttagaa ctgtatctat tgtttgcgga gcgtttgccc 1980
gttcctgacg tcgcgttccg tccttcagcg gacgttgtcg ttttctggcg atcacagcta 2040
caacgctatc gagtgttgcg tgattccttt tctaatgtgc cctccatcgg gtccactctc 2100
actttagatg aacctttgac tgtctctatt ctcaattttg ttgacgtcac ctccctttct 2160
tctcttgagg atcaacgagc cttatctgct ttttcagttt taacttctct agggtcacag 2220
aaactctcgc ttcatcctta ctttgatagc taccgcacgc agctcactgg aataattact 2280
ccacattcac gtaatcttct agatagactg gcgtacgtcc cgcgcgtttt tccttcgacg 2340
attgatgtgc aacatcgtgt catggcctct tcagatccag aaatttttgg ttttcgttct 2400
aattcatgga ctcaactgtc cttcttctac gacgcgacgt taacttctat tgattttact 2460
gatgtaaagc actggttaga tttagggacc gggcctgagg cgcgaccgtt gtcttttctg 2520
ccttccgatc ttcccgtcac gttatgtgac actcgtccat tcatcttccc ttccggctgc 2580
tgggctactt tcactgattt cttaagttat gactaccttg tcacgaatgt cgtgctctca 2640
actggtgccg atgtcgtatc ctgtgttctt tctctgggtg cggcctgtgc tgatgccaac 2700
ataactttac atgaaggcgt gcggcagctg atttcccaat gcgtggatgc caatgtcaag 2760
acattgttcc tgcagcttaa ttgtcctctc ccatctgcgg gtgatgtatc tcgggagatt 2820
cttgagatgg ttcagactaa ttcaacttac gtgtttcata ccttgggtcg tattgaaccg 2880
ttcattccat actccgctct ttcagagata gttgaggact tgtgtcccgg catcgtcatt 2940
gaaattaaga ctatggatcc ctctctctca tggcttgatt acgctgttca atccaatgcg 3000
tcagtgacgt cggatgatat cgtattggca atgcgtctgt ctcacttctg tccacttttc 3060
gtgtttcatt ttgaccgtca gtctgctcaa tttccggatg atgcgcgtgt tgggactcct 3120
tttactgtca cgctgttaga ttatgaagat actcgttcat acgaggtgac gttagataat 3180
gtcactatcg ccaccgttac cgcaggtgct ttggtgggtt tctcatctgg tgtcagcgtc 3240
agttcaacca acaatcagct tattttaact atcgattccg caagtccagg aattctctcg 3300
gtcattcaga ttcttcccgc tcgtatctct ttaggcagtt gtgtgataga agcaccggat 3360
ccatctctct ccttgatctt ccccgccacg ttagatacct ctttgtcggg aaccgatttg 3420
gagctgtatt tgtctgactg gtacgatgtt gcattatttt acgtcgatga aatccactcc 3480
cgcttgctgc cagtgtccga taccaagtat gaaatatatc gcaaggatca ggcgccgaac 3540
agccgggtga tcaactatat tttcgatcgg tctgatgtgt ttttcaagct agtgttatgt 3600
gacgtatctc cctcaggagt aggccgtttc atctaccgtg agttgccaga attaagttca 3660
cctgtttggc cagacaacgc gcgcactttc ttgtccatac cgttcgagtc acccatggtg 3720
attgtctcgc cggacggacc tgttaattac gatggcgcaa actttactcc tccaaactca 3780
tggttgacgg ttgatggcag tacctgcgtt gtagatggcc gtccttcgtt ctacgtgccc 3840
cctggccgat atggtctggt gagagtctaa acgacc 3876
<210> 6
<211> 2199
<212> DNA
<213>M1 genetic fragments
<400> 6
atggcctatc tagccacacc tgtgctagga gtcggttctc gcattaccgc tttagatcgt 60
actattgatg ctatcacgtt gaaacctcga atcgacttac aagatgtata cacaattgat 120
cccactttga ctctgcgtca gatagagtta atctcttcgg gaacttcaat ggacgatatc 180
gctcgtggac tgttgcaccg agactggcgt cgtcaatcca tcatcgtttt gcttccctcg 240
cgtcgctctc tccttgagta tctattgtct aacccttctg tctgtccaga cggtttagat 300
cgttctcgac ttaaaggatt tcagaagcgt ccaaatgatt ttcgtgttca agatttcttc 360
tctccactga tcacggactc gacgtcaatt gctacatact ctcgatggct taatgcccac 420
cctgttgtgt actcaactac tcataaggtc gctggtgctc gggtgcgtct ctttggacct 480
gccaaattat acattctgtc acctgacgtt cttcgcgaat tatccatttt gagatccacg 540
gatcgtgtcc tcgttgtacc tacagcacgt gtatatgttg gttgctttcc tagcgcttcc 600
actagtaatt gtgtgctcac tgcacgcgac cgctggaatg ctcctgacgt tcatcccgtt 660
gtcaaggcaa tccaattagc atatgaccat caatatcgtg tcaccgctcg ctatctttcg 720
gatccccttg tctccgccct tcttgttggg aatcggtcgg tgaagacctt gaaggtacag 780
ccagtagagg ccagagcagc acgatcagtc ggcattcgcg ttcaagcgat gacgccccct 840
cgtggtatca acacctctat catccaagtc gttgatctca ggctgcaatg tcgacattct 900
ctcattccca ccgaaaggcc cttcccgctg acatttatcg gcctcccatc ctgtttgctc 960
cagcatttgg atttgacgct atctgacgat tgggtgccta ttcgtgatcc cacgggcatg 1020
tttgaaatgt ggttcatgat tcttacgctc acttgtgata agattcttga tggacggggc 1080
aacgctgttt ttctcatccc cagttctact aatgcattgt cgattaatta tgtacagctt 1140
acatcgaccg cgtctcaacg ccctcagtca ttagcggcaa atgcatctgg acggatagat 1200
tctatcggac tatgtatgcc taaggggtct tttaagtcaa ctatgattaa atttctcact 1260
ggcttggaga tttgcggcac acgagtgatg tactcggacg tcgtgatgga cagtgatgac 1320
gtgggtgacg ctttggatcc tacttttgaa acggctttgt atgatgcact ggtagcactt 1380
gatccgcctt ttgacgttga taagttggct agccccactg atctagttaa tcaggagtac 1440
gttgcgtctc atatgtaccc gacattctta cggcttgtca atgagctgct gactcctaag 1500
gcttcagagt tgtactctga gcgtagcgtt gaattccgat ctcttactta cgcgcacgct 1560
gattctgaat ttcttaactc atgctggacc gctcgcttaa tgcgttgctt tatcaactat 1620
catgaagagc agaatatctt acttcgtcct ggacgcgttg gtggggtgtt atttcaagtc 1680
gcgttgagcc gttgctataa gatgttcgct acttccactc ctgcttcccc tctgtcattg 1740
ttcctcaagt cgttgttcgt tccttggatt gagtctgccc cactgttagc gaatctaacg 1800
ccaaatgagt cttctcgtgt gttagcatgg tatattcctt cctcgtactg gagcgacaat 1860
ggttggtgcg tttgtgacac tcatcgtcac gtcaccttct ccttcatccg cggtcttccc 1920
gccgacctgt cggtgttaga tctgtttgat tggtctcgat tccgcgcgac tataaacgtg 1980
gacacgtctc tcgtggagct aggcgcagac attcgtgcgg tcaaagtatc agtccattgg 2040
acatctcaga agcccactgt ggacgttttt gacaatcgtg cgcttttcac ccccttccag 2100
cactaccatt tgagtctcca ctgtaattgc gcacctggtc gacctttctt cgcgaagaac 2160
atgaagctat atttgtcgac ggtaggaggc gagcactga 2199
<210> 7
<211> 2208
<212> DNA
<213>M2 genetic fragments
<400> 7
accaccgaga tctacactct ttccctacac gacgctcttc cgatctcttt ctcacaagat 60
gggtaacgcg acgtctgttg tgcagaactt caatatccaa ggtgatggta atcattttgc 120
tccatctgct gagactactt catccgccgt accgtcatta tctttgaatc ccggactgtt 180
aaatccaggt ggtaaggcgt gggtcctgat tgatccatct ctaaatgctt ccgatccttc 240
atcactacgt ctgatgactt cggctgatct atcaacactt cctcgatctg ctactagtaa 300
ctctaccggg tttctcccca cttctggcat gtatgccatt gctactaagg agacgttgag 360
tgtaattact gagcacgcga tttcccagtt tgataagtta cagatggctt gtgagttgga 420
ccgcgattat ctggatgcta gaggtgtttc tcctgagtct gtgaatattc atagttatat 480
agcctacgtt gattgcttcg tgggtgtatc tgcaaggcag gctgcgtcaa attttaagcg 540
gcatgtgcca gttatcacca aatctcgtat gacacaattt atgacatccg cgcagaatat 600
gttgcaagtg cttgggccct gggaacgtga tgttcgtgag ttactcacta ttcttcctac 660
ttccactacc gctggtaaaa ttacgtgcga catgaagtct gttgtcgctt tcattgatga 720
tcagctctct gataccagtt tgtgtcgtct gtaccccgac tgtgctgctg cggcggtggc 780
tagacgtaat ggtggcattc gatggaagac acctgatact gacgaggctc cttcacttgc 840
aactaacgat attgctgctt caactatggg tacgcttgcg aatactacac cactggctga 900
gaagtcgaac tcgggcgagg agtcgatgcg cttggttagt gatgttggcg tggacatcgt 960
ttgttctcgt ggccccatca gttcttcagt ttggtcccgc acggttgaac ccaaatcgta 1020
caatattaga acccttcgtg tagaagaagc gctttggcta cgcgagtgcc aagcgactac 1080
tggttttgat gtacagtaca cgctgcccga ccagactaca cataagcatt tctggcttca 1140
gaaggggtca gtcgtcataa atcttgagca aacgggtagt atgatgttcg atgtgaacat 1200
agcgggtaaa gattacaaga agggcacctt taatcctgat aatcataaat tggtcctctt 1260
ggttatgcag tcaaagatcc ctttcgagtc ttggaccgtc gcttctcaaa ttactggtat 1320
cgctcaagtg gctgaggtca ctgtgcatgc tgctgatagt tcgactccta accaaaagat 1380
aatcggtgaa acttcgctgt cttatttatt tgagagggag acggtgacca catccaatac 1440
tgaagtcaat acatatctgt tgtgcacttg gcagcttgac gacgcgcaga gcaatgacgc 1500
aaacgcctgg ccagatgctt gggacgggat cacaacattg accccactta cgtccggtac 1560
tgtaaccatc aaggggactt cggtggattc tgtcgtaccg tctgatttag ttggtgctta 1620
tacacctgag gctttggctg ccgcgcttcc taacgacgct gggttaattt tggctaataa 1680
ggcaactaaa ttggctgacg ccatcaataa ggaggatgat tctgtgattg atgagtcttc 1740
tccctttagc acccccattc aaggagttct ggctgttcaa caacttgata ccgtggggac 1800
acgcggtaca cgtgcactcc agcctccatc cattctgaaa cgcatcgcct cacgagctct 1860
tcacatgttt cttggtgatc caaagtctat tctaaaacag gcgacgcccg tattgaggga 1920
ccctgacgtt tggaccggct ttgttcaagg tgttagagac ggcatccgga ctaagtcgct 1980
atccgctgga gtacggtccg tgtataataa cgttaccgcc acacagtctg tacaaacgtg 2040
gaaacagggg ttcctgacga aaatacagac gttgttcaag ccatcgtgag gtgctaaggc 2100
ctctctctgc ggcgggtcgg tgggcacgtc gtagtgacgc tgaatgcacg gggaggtgac 2160
gctccctgga ttggcaagat cggaagagcg tcgtgtaggg aaagagtg 2208
<210> 8
<211> 1996
<212> DNA
<213>M3 genetic fragments
<400> 8
gctttttgag tcctagcgtg gatcatggcg tcaaccaagt ggggagacaa gccgatgtcg 60
ctctcaatgt ctcacgatgg atcatctatc cgcagtgccg cctcacagtt tctgtcggtt 120
cccctgtctc actcaacgcc aatcccacct caacggaaga ccgtattgct gaaattcatg 180
atcggtgatg acctggttac cgtgcagggc gcgctcgctc cttttgatga atactggtac 240
gataaccaac ctctattgtc tcaagctgtt gagctgctcg cctctgagga tcgtctgcgt 300
caattcgagc attatgagaa gtttctactt aagaagggcc accaaattgc tgagatcatg 360
aataggctac gtcttttctt caccgacgtt ctcaaagtga agatggaagc tgatgctctt 420
ccttctctag ctcaatacct gatggctggt actttggatg ctgtctccac cgttcacgaa 480
cctgatgctt gtgttccagt cacttcaaaa atcatagcta agcagcagac tgtgtccaag 540
tcccctggac gtcttgctga agaggagtat aatgttatta gatcacgttt cctcactcat 600
gagatctttg acttaacgtc tgacttgccc ggtgttcaac cattcatgga tatgtactat 660
gccaccgttc ctcgtgccga ttccaccggt tggtgtgtct accgcagaaa aggtctattg 720
attcatgccc ctgatgagca atactcggat ctgactattt tcaccacccg tcttacggca 780
gcgcgtgagt tacagcttgt ggctggggag gtcgttgtgg cttgcttcga tcttatggat 840
atctctgata ttgctccatc tcatcatgca tcggttcaag aggaacgtac tctcggcact 900
agcaagtatt ccaacgttac agctaatgag catccgttgg tattcttttc acccaatgca 960
ttacgctggg caatagatca tgcctgtact gattccttga tttccactag gaatattcgg 1020
gtctgcgtcg gtattgaccc cttagtgacc agatggactc gcgatggcgt gcaggaggct 1080
gcaattctta tggatgacaa gctaccctca gcaggacgtg ctcggatggc tctacgaacc 1140
ttgcttctag cgcgtcgctc accaatgacg tccttcttac taggtgctct caagcagtcc 1200
ggtggtcagc taatggaaca ttatcgatgt gatgcggcta ataggtatgg atctcccacc 1260
attccagttt ctcaccctcc accgtgtcca aaatgtcctg agctgaaaga acagatcacc 1320
aaactttcgt cagctcctgc gcctaaagtt gactcgtccg ctggtcctgc cgtgctgttg 1380
tcgaagatcg ctgagctcca acgtgctaac cgagaactgt ccttgaagtt agtggacgtt 1440
cagccagccc gagaagacca ccttctagct tacctcaacg agcacgtatg tgttaacgct 1500
aaagatcacg agaaaggtct actagcccgt tgtaatgtct cgagtgattc aatcgccgct 1560
atccttggtc aacgtttgaa aaatcgagaa cggtttgaaa cgagactacg gcacgaggct 1620
ggtgcggagt gggagccacg agtggaagcg ttgaatcaag agttggctaa ggcgcgtatt 1680
gagcaacaag atatgatgac tcagtcctta cagtacctga atgaacgtga tgaactgctg 1740
cgcgaggtgg atgagctcaa acgcgaactg gctaccctac ggtttgctaa tgtgagacta 1800
aatgccgata accaccgcat gagtcgtgcg acccgtgttg gagatgtatt cgtcagtgat 1860
gttgatccct taccacccgg tcttcctggt gaatcgaaac catccattga agaactggta 1920
gatgatctgt gagctttgcc ttgtgactcg acttctctct gattccatgt acccacggcg 1980
gactcggcta ttcatc 1996
<210> 9
<211> 1644
<212> DNA
<213>S1 genetic fragments
<400> 9
gctttttcag tctcttgtat cgatgttccg tatgtcttcc ggttcatgca acggcgcgac 60
gtcaatattt ggtaacgtgc attgtcaagc ggcccaaaat accgcaggcg gagatcttca 120
agctacctcg tcattaattg cttattggcc ttatctcgct gctggtggtg gtctcattat 180
aattttaatt attgttatag gtataatctg ttgttgtaag gccaaagtta aggctgacgc 240
taccagaagt gtgttctatc gggagttgct tgctctgaac tcgggcaagt gtaatgcagc 300
acctccgtca tacgacgttt gatgtgcggc ggtttgagtt ttctccgacg gtgtttgaag 360
agtgtttgac tccatctttt accgctgtga ctgacactga ccctgtgagg tactttaata 420
ttgagcttcc gtcaactcac cgtctcctcc cttggcttcc cgttcttctg ttccaatcct 480
gtaaagtgca tgtttcttta gtacgtagat tctctttatg ctcgacttta tctgatattt 540
gtgagtacga ttgcaaattg cttccgtcta ttaacgctat tgtgtcgaat ccagtgtcga 600
gcgcggtttc atctatcgtc gttcactggg atggacggat taactcaaca gcagcgaaga 660
gaagtcgtgg ggttgatact gtcgttgact tcgagcgtga gtataagttc tggcgatttg 720
acgcaaattc gtgagcgtct ttccgctttg gaatctgcga ctgcgtcgct gaacgaatcc 780
gttaatacag ctttgtctag gttagtggat ttgtctgcat cgcttgataa cgtggcggcc 840
tcgttagcgg agacgaaagt ggaaatgaac tcactagttt ctgacgttca gggtttgcga 900
gcttcccttg actcttctgc ttcagagctg gcttctctat cttcattggt gcgtgatcac 960
ggctcttcga ttgctggcct acagagagaa gtaagtgcct tatcgagtga ggtaggcaac 1020
cttaaaacct cggtatcatc gcagggcctt cctatcacta gccttgagaa acgagtgcaa 1080
gctttagaag gtggttctag tacgactctg tcatttgctg atcctcttaa gttagaggct 1140
gggaccgtgt cactcgaggt agatccgtat ttctgctctg tgaatcgtaa tctgacgtcg 1200
tattctgctg atgctcagtt gatgcaattt cagtggtctg tgaaagggga agatggcgcg 1260
gccaactcta ttgatatgga cgtgaacgct cactctcatg gttcacgcac tgattatctg 1320
atgtcaacca agcaatcatt gactgttaca acgtctcccg ctactcttgt ctttgaactg 1380
gataggatta ttgctcttcc ctccgacctt tctcgcctaa ttccatgtta tggttttcag 1440
caagccactt ttcccgttga tatctccttc cagcgagatg gcgtttcgca tacgtatcaa 1500
gtctatggga agtacacatc ttctcgcgtc ttcaagacta cgttctcgcc tggctcctca 1560
ggtcccgcag tgattaagtt tttgaccgtg cgtacgggca tcgataccta aggtgtggcg 1620
ccgtacgggg gctggttatt catc 1644
<210> 10
<211> 1322
<212> DNA
<213>S2 genetic fragments
<400> 10
gggcgcgtgc cgtgtacgac ttcttttcta cgcctttcgg gaatcgtggt ctagcgacga 60
atcgtactca actatcatca ctactatcaa gctcgaattc cccatggcaa cgttttctat 120
catcaatgac tccattgaca gcgccgggca tcgtttcgac acctgaagca ccctatccag 180
gttcgttaat gtatcaagag tctatgctcc acagtgctac cgtccctgga gtacttggca 240
atcgcgacgc ttggcgtacg ttcaatgtct tcggactttc atggactgac gaaggactgt 300
caggactagt ggctacccaa gatcctcctc ccgccgcccc gtatcagcca gcctctgctc 360
agtggtcgga tcttctcaac taccccagat gggcaaacag acgtcgtgag ctgcaatcta 420
agtacccgct tctgcttcgc tccactctgc tctctgccat gcgagctggt cctgttctat 480
atgttgagac gtggccgaat atgatttctg gacgattagc tgattggttt atgtcccaat 540
atggcaataa tttcgttgac atgtgtgcta ggttgaccca gtcttgttcg aacatgcctg 600
ttgaacctga tgggaattat gatcaacaga tgcgtgcttt aattagtttg tggcttctgt 660
catacattgg ggtaatcaac caaaccaaca ccatcagcgg tttctacttc tcctcaaaga 720
ctcggggtca agcgttggac agttggactt tgttctatac cacgaatact aatcgtgtcc 780
aaattacgca gagacatttt gcttatgtgt gcgcccgatc tcctgattgg aacgtggaca 840
aatcatggat cgctgctgcg aacttaaccg ccattgttat ggcttgccgt caaccgccga 900
tgtttgctaa tcaaggcgtc attaatcagg cgcagaaccg acccggattc tccatgaatg 960
gggggacgcc cgtccacgag ctcaacttac ttactactgc gcaagagtgc atcaggcagt 1020
gggtggtagc aggcttggtg tcggcagcaa aggggcaagc actaacgcag gaagctaatg 1080
acttctcaaa cctcatccag gcggatctag gccagatcaa ggcgcaggac gacgctttgt 1140
acaatcagca gccgggatac gcgaggagaa taaaaccttt cgttaatggt gactggacac 1200
caggtatgac cgctcaagct ctggccgttc tagccacttt taccgcctag gcgtagggtc 1260
gtacgctgcc cgagtccagc cctccggcag cccgtggaga tcggacgagc gtcgtgtagg 1320
ga 1322
<210> 11
<211> 1202
<212> DNA
<213>S3 genetic fragments
<400> 11
gctttttgag tccttagcgt gcaagccgca atggaggtac gtgtgccaaa ctttcactcg 60
ttcgttgaag gaataacatc tagctacttg aagactcctg cttgctggaa tgcacaaaca 120
gcttgggata ctgtgacctt tcacgtccct gatgtaatta gagttggtaa cgcctactgt 180
tgttctcaat gttgtggtgt actctactac gggactctgc cctcggacgg taattatttc 240
cctcatcaca agtgtcatca gcaacagttt aggactgata ctccactgct tcgatacgtg 300
cgcattggta gaaccactga gcatctgatg gatcaatatg ctgtcgctct ggagtccatt 360
gctgaacact atgacgagat tagtcaacgt atggtcgatg agccagagaa tgacgaggtt 420
acacctcttg atatcgttac gcgtaccgaa tctatcagga gtgacaaggc agtcgaccca 480
gacttttgga catacccact tgagcggcgt tctgatgatt ctcgtagaga catcgcctca 540
gcatgctgga aaatgattga cgcgtcggcg cgtagtctca ctcttccaaa ttgcctcgtc 600
tccccctctt tgcactctcg ctccgtcttt ggtcagatgc aaacgaccac cactatatac 660
gatgttgcgg catcgggaaa ggccgttaag ttttcaccaa tggttgctac actagcgcaa 720
cgtgatgctg gccctgtaat gcttgcgaat gctgacccgg cggaaggcgt gtactctttc 780
tggacgtcgc acttcgcttt ctcaccgctc atcggcggag ttggaattac gggacagtac 840
gctcgtgagt cgtaccatca agtgggtcat ccagtgattg ggagtggtaa gaaggcatcg 900
cattacagga atctgttcat ggaagcgtgg cgcgggtggt cgaagtcagc tttcgcatgt 960
gctactggaa tggagccagc tgaatgtgaa tctcgtctga gaggacacgc tcgtactatg 1020
ctcggacgct ctctgccgcc cgtttgtgac gatgatgttg ctcagcagtc tggcgcggta 1080
ctgacttcgc tgcagaaaac gaacaagttc accgttgtgg agtgtggttg gtaagtacct 1140
ccgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggact cgcctattca 1200
tc 1202
<210> 12
<211> 1192
<212> DNA
<213>S4 genetic fragments
<400> 12
gctttttgag tccttgcgca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60
catccggcgc agctggtcag actgttttta gaaactttta cttactacga tgcaatatct 120
cagctgacgg tcgtaatgca acaaaagctg tgcaatccca ttttccattc ctttctcgtg 180
ctgtccgttg cctatctcct ctagccgctc attgtgctga taggactctt cgtcgtgaca 240
atgtgaaaca aattcttact cgtgagctgc catttccatc ggatttaatc aattacgcac 300
atcatgtgaa ctcatcctcc cttactactt ctcagggtgt cgaggcggca cgtctagtgg 360
cccaagtcta tggagaacag ctatcgtttg atcacattta tcccactggt tccgcaactt 420
actgccctgg agcgattgct aatgcgattt cccgtatcat ggctggtttt gtgccccacg 480
aaggtgacaa ctttaccccg gacggttcta ttgactatct cgccgccgac ctggtcgcgt 540
ataagttcgt gctcccttac atgctagata ttgtggacgg acgtccgcag attgttcttc 600
catcacacac tgttgaggag atgctgtcca acacgagttt gcttaattcg attgacgctt 660
catttggtat tgaatcgaag agcgatcaac gcatgacccg tgacgcggct gaaatgagtt 720
ctcgctcact taatgagctt gaggatcatg agcagagggg tcgaatgcct tggaaaatca 780
tgacggcaat gttcgcggcg caattgaagg tggagttgga cgccctagct gatgagcgcg 840
ttgaatctca ggctaacgct catgtgacat cttttgggtc tcgtctgttc aaccaaatgt 900
ctgcttttgt cccaattgat cgtgagttga tggagctggc tctactcatc aaagagcagg 960
gtttcgcaat gaatccaggg caagtcgcat ctaaatggtc gctgatacga cgatctggcc 1020
ccactcgccc gctatcaggc gcacgccttg agatcaggaa tggcaactgg acaattcgtg 1080
aaggtgacca gacgcttctg tctgtctccc cagctaggat ggcgtaaacg ggacccatgg 1140
tgcgggtgag gggccgccac accctctgcc gcgacctgga ctcttattca tc 1192
Claims (5)
1. one plant of duck reovirus, deposit number are:CCTCC NO:V201818.
2. duck reovirus described in claim 1 is preparing the application prevented during duck exhales the vaccine of the lonely disease of intestines.
3. application according to claim 2, which is characterized in that the vaccine is inactivated vaccine or attenuated live vaccine.
4. application according to claim 3, which is characterized in that the vaccine is inactivated vaccine.
5. duck reovirus described in claim 1 is in the application in preparing treatment duck to exhale the Yolk antibody of the lonely disease of intestines.
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CN109718370A (en) * | 2019-02-27 | 2019-05-07 | 广东渔跃生物技术有限公司 | A kind of duck reovirus vaccine and preparation method thereof |
CN111172122A (en) * | 2020-01-07 | 2020-05-19 | 齐鲁动物保健品有限公司 | Duck reovirus attenuated strain and application thereof |
CN113493507A (en) * | 2020-12-31 | 2021-10-12 | 哈药集团生物疫苗有限公司 | Novel duck reovirus egg yolk antibody and preparation method and application thereof |
CN114592088A (en) * | 2022-02-16 | 2022-06-07 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | Multiplex PCR kit for detecting or distinguishing 4 different genotypes of avian reovirus and application thereof |
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CN109718370A (en) * | 2019-02-27 | 2019-05-07 | 广东渔跃生物技术有限公司 | A kind of duck reovirus vaccine and preparation method thereof |
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CN111172122A (en) * | 2020-01-07 | 2020-05-19 | 齐鲁动物保健品有限公司 | Duck reovirus attenuated strain and application thereof |
CN111172122B (en) * | 2020-01-07 | 2022-07-12 | 齐鲁动物保健品有限公司 | Duck reovirus attenuated strain and application thereof |
CN113493507A (en) * | 2020-12-31 | 2021-10-12 | 哈药集团生物疫苗有限公司 | Novel duck reovirus egg yolk antibody and preparation method and application thereof |
CN113493507B (en) * | 2020-12-31 | 2023-04-21 | 哈药集团生物疫苗有限公司 | Novel duck reovirus yolk antibody and preparation method and application thereof |
CN114592088A (en) * | 2022-02-16 | 2022-06-07 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | Multiplex PCR kit for detecting or distinguishing 4 different genotypes of avian reovirus and application thereof |
CN114592088B (en) * | 2022-02-16 | 2024-04-02 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | Multiplex PCR (polymerase chain reaction) kit for detecting or distinguishing 4 different genotypes of avian reovirus and application thereof |
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