CN108754019A - A kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences - Google Patents
A kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences Download PDFInfo
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- CN108754019A CN108754019A CN201810532731.9A CN201810532731A CN108754019A CN 108754019 A CN108754019 A CN 108754019A CN 201810532731 A CN201810532731 A CN 201810532731A CN 108754019 A CN108754019 A CN 108754019A
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- orf1
- diarrhea virus
- epidemic diarrhea
- porcine epidemic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences, 8 pairs of primers involved in amplification method, each primer pair upstream and downstream primer is successively such as NO.1~16 SEQ ID.Specific amplification step is extraction PEDV total serum IgEs, is inverted to cDNA, is carried out amplification reaction with 8 pairs of primers, PCR product is connected in cloning vector pTA2 Vector, TOP10 competent cells are converted, chooses positive colony, sequencing identification, finally splices sequence, obtains ORF1 genom sequences.The ORF1 genes for obtaining Porcine epidemic diarrhea virus variant in pathological material of disease can be expanded by this method, and can capture the variation of Porcine epidemic diarrhea virus variant gene order during cell culture passages.A kind of method detecting PEDV in pathological material of disease is can also be used as, and is laid the foundation for the research of the ORF1 gene-correlations of PEDV.
Description
Technical field
The present invention relates to biotechnologies, more particularly, to a kind of Porcine epidemic diarrhea virus ORF1 gene total orders
The amplification method of row.
Background technology
Porcine epidemic diarrhea virus（Porcine epidemic diarrhea virus, PEDV）It is pig epidemic diarrhea
（PED）Cause of disease for a kind of single-stranded positive RNA viruses for having a cyst membrane be the member of coronaviridae α coronavirus genus.It should
Disease takes place mostly in cold wave season, and infection piglet can vomit because of watery diarrhea, dehydration and electrolyte disturbance and death.It is fallen ill
Severity and the age in days of the death rate and pig it is negatively correlated.Especially for the newborn piglet in 7 ages in days, lacking effective source of parents
Under antibody, the death rate may be up to 100%.It based on the appearance of popular street strain in recent years and is very popular, traditional commodities vaccine is to working as
Preceding prevalence street strain, which lacks, generates sufficient resistant function, and high mortality keeps pig breeding industry economic loss heavy.
1971, PED was reported for the first time in Britain.Many countries in Europe report that the prevalence of this disease is broken out successively.Billy
When scientist class coronavirus CV777 was isolated from the diarrhea pig for breaking out PED in 1976.2005, Chinese scholar
It is separated to one plant of wild poison, is named as LJB/03.2006, Chinese scholar was separated to 6 plants of open countries from the class in piglet abdomen gulf just sample
Poison.At the beginning of 2006, PEDV epidemic situations start outburst swinery after immune.2010, there is epidemic situation successively in the major province of China.2010
In October in year, large-scale PEDV infection has been broken out in domestic southern several provinces, and travels to other areas rapidly, especially
It is the Northwest.2011~2012 years, the major province of China large-scale outbreak PEDV epidemic situations.
Invention content
The purpose of the present invention is to provide a kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences, the party
Method can expand the overall length of Porcine epidemic diarrhea virus ORF1 genes, the kind poison for the different generations especially cultivated on cell
ORF1 gene complete sequences.Porcine epidemic diarrhea virus can be captured during cell culture passages by this method
The sequence variation of ORF1 genes, to by the Genetic Variation Analysis of gene understand its to virulence or immune efficacy it is related
Property.
To achieve the goals above, the present invention is achieved by following scheme：
A kind of amplimer group of Porcine epidemic diarrhea virus ORF1 gene complete sequences, is made of 8 primer pairs, in each primer pair
Downstream primer is successively as shown in NO.1~16 SEQ ID.
A kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences, includes the following steps, extraction pig is popular
Property diarrhea virus sample total serum IgE, be inverted to cDNA, be utilized respectively 8 primer pairs as described above and carry out pcr amplification reaction, respectively
Cloning and sequencing is carried out, finally splices sequence, obtains ORF1 gene complete sequences.This method amplification target fragment ORF1 overall lengths be
20345bp, by being respectively 12,354 nucleotide and 8 comprising 46 nucleotide overlaps and length, the two of 037 nucleotide
A big reading frame composition, occupies genome nucleotide quantity about 2/3.
Preferably, the program of the pcr amplification reaction is 94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 10s；It is moved back under annealing temperature
Fiery 30s；68 DEG C of extension 2min；Cycle 35 times, 72 DEG C extend 10min eventually；It is respectively right that the annealing temperature corresponds to each primer pair
The annealing temperature answered.
Preferably, 8 primer pairs are respectively primer pair ORF1-1, ORF1-2, ORF1-3, ORF1-4, ORF1-5, ORF1-
6, ORF1-7, ORF1-8, each primer pair upstream and downstream primer is successively as shown in SEQ ID NO.1~SEQ ID NO.16.
Preferably, the annealing temperature of 8 primer pairs is respectively：
Primer pair ORF1-1, primer pair ORF1-6, primer pair ORF1-7, primer pair ORF1-8, annealing temperature are 62.5 DEG C.
The annealing of primer pair ORF1-2, primer pair ORF1-3, primer pair ORF1-4, primer pair ORF1-5
Temperature is 65 DEG C.
Preferably, in the above method, the specific method of the cloning and sequencing is by amplified production recovery purifying, amplified production
3 ' ends add base A, are then attached in cloning vector pTA2 Vector, convert TOP10 competent cells, screening and identification
After recombinant bacterium, it is sequenced.
It is described splicing sequence specific method be：Sequencing result utilizes in 7.10 bioinformatics softwares of Lasergene
Editseq softwares are removed the export-oriented terminal sequence including primer pair, the splicing of full length sequence and homology analysis simultaneously,
Tools BLAST is quickly compared with GenBank in NCBI to carry out the verification of sequencing result or compare to search for, and completes sequence and data
Existing sequence in library is compared.
The application of above-mentioned primer sets or/and amplification method in terms of Porcine epidemic diarrhea virus ORF1 gene complete sequence amplifications
Also in the scope of the present invention.
The present invention also provides a kind of method of specific region on amplification Porcine epidemic diarrhea virus ORF1 gene complete sequences, institutes
It is to be carried out amplification reaction according to arbitrary above-mentioned 8 primer pairs of combination of needs to state method.
Application of the above-mentioned amplification method on Porcine epidemic diarrhea virus ORF1 gene complete sequences specific region is studied also exists
In the scope of the present invention.
Above-mentioned amplification method can obtain the ORF1 genes of Porcine epidemic diarrhea virus variant in pathological material of disease.To capture pig stream
The variation of row diarrhea virus variant gene order during cell culture passages.It can also be used as in a kind of detection pathological material of disease
The method of PEDV, and lay the foundation for the research of the ORF1 gene-correlations of PEDV.
Compared with prior art, the invention has the advantages that：
Specific designs of the present invention go out one group can clone Porcine epidemic diarrhea virus strain passage the 65th generation after ORF3 genes it is complete
Sequence.The primer sets provided through the invention can expand the ORF1 bases for obtaining Porcine epidemic diarrhea virus variant in pathological material of disease
Cause, and the variation of Porcine epidemic diarrhea virus variant gene order during cell culture passages can be captured, it can also
As the method for PEDV in detection pathological material of disease a kind of, and lay the foundation for the research of the ORF1 gene-correlations of PEDV.
Description of the drawings
Fig. 1 be the present invention design to primer amplification result agarose gel electrophoresis figure；M is the Marker of DL5000；Swimming
Road 1~8 is respectively：ORF1-1,ORF1-2,ORF1-3,ORF1-4,ORF1-5,ORF1-6,ORF1-7,ORF1-8.
Fig. 2 is position of the ORF1 genes in PEDV genomes.
Specific implementation mode
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
A kind of Porcine epidemic diarrhea virus ORF1 genes complete sequence amplification method, comprises the following specific steps that：
1, design of primers and synthesis：The nucleotide of Porcine epidemic diarrhea virus variation pnca gene disclosed in GenBank
Sequence, design primer obtain the primer sequence of following sequence, primer sequence and each primer sets amplification length by largely screening
As shown in table 1.
Table 1 is the amplification length of the sequence and gene of 8 primer pairs
2, the extraction of virus total RNA：The isolated popular strain in inventor laboratory is passaged to the disease in 65 generations on cell
250 μ L of malicious culture solution are added in the Trizol of 750 μ L, take the PBS of 250 μ L in a sterile centrifugation tube, 750 μ L are added
TRIzol acutely vibrates, and acts on 5 minutes at room temperature.Again plus 200 μ L chloroforms, acutely oscillation are placed 5 minutes at room temperature.4℃
12,000 r/min are centrifuged 15 minutes.500 μ L upper strata aqueous phases are moved on in another new sterilizing microcentrifugal tube, 500 μ are added
L isopropanols, soft mixing, -20 DEG C are placed 15 minutes.12,000 r/min are centrifuged 15 minutes, and incline supernatant.It is added 1,000
75% ethyl alcohol of μ L ice precooling, it is slowly reverse to shake up.4 DEG C of 12,000 r/min is centrifuged 15 minutes.Gently discard supernatant, placement 3 ~
5 minutes to air-dry precipitation.The RNase Free distilled waters dissolving RNA of 20 μ L is added, is saved backup in -20 DEG C.
3, the synthesis of virus the first chains of cDNA
It is spun with reference to Japan（Shanghai）The improved high-efficiency Reverse Transcriptase kit ReverTra Ace of bio tech ltd
（TRT-101）Specification carry out, obtained cDNA is placed in -20 DEG C of preservations.
4, the amplification of PCR
With 8 primer pairs, expanded using following reaction system and response procedures：
The system of the amplified reaction be 10 μM of 1.0 μ L of sense primer, 10 μM of 1.0 μ L of downstream primer, 2 I-5 ×
High Fidelity Master Mix 12.5 µL、ddH2O 8.5 µL。
The program of the amplified reaction is 94 DEG C of pre-degeneration 2min；94 DEG C of denaturation 10s；Anneal 30s under annealing temperature；68℃
Extend 2min；Cycle 35 times, 72 DEG C extend 10min eventually；The annealing temperature corresponds to the corresponding annealing temperature of each primer pair
Degree.
5, the solidifying electrophoresis of amplified production
（1）Ago-Gel preparation weighs 2.0 g agar Icing Sugar and sets in conical flask, and the electrophoresis liquid of 100 mL is added, is made into 2%
Ago-Gel makes agar Icing Sugar fully dissolve in microwave stove heat.It then takes out to be placed in room temperature sink and is cooled to 50 DEG C
Left and right is added 7 μ L Gold View nucleic acid dyes, mixes well.Gel processed, at room temperature about solidifiable after twenty minutes.
（2）Nucleic acid electrophoresis adds to PCR product in the hole of gel, separately adds 5 μ L DNA Marker DL5,000 opposes
According to.The electrophoresis 30 ~ 40 minutes under 120 V voltages.Result and preservation of taking pictures are observed in gel images processing system.
6, the recycling and purifying of amplified production：It is of short duration in ultraviolet lamp by all amplified productions into row agarose gel electrophoresis
Target fragment is cut under irradiation, and recovery purifying is carried out with reference to the quick QIAquick Gel Extraction Kits of Magen company DNA.
7,3 ' end of glue recovery product adds base A：It is flat end since high fidelity enzyme amplifies the segment come, and normal enzyme
With the characteristic for adding a base A in 3 ' end of product, it is possible thereby to the pTA2 Vector carriers with cohesive end are connect, from
And the efficiency that segment is connect with pTA2 Vector carriers can be greatly improved.Specific reaction system is shown in Table 2.
Table 2 is that 3 ' end of glue recovery product adds base A reaction systems：
Response procedures are 72 DEG C, 30 minutes.
8, target fragment and carrier connect：By 3 ' ends add base A product carry out concentration mensuration, with the amount of 125ng with
Carrier is attached, and linked system is shown in Table 3.
Table 3 is linked system：
By reaction solution in room temperature（15~25 DEG C）React within 30 minutes.
9, connection product transformed competence colibacillus cell：By Competent Cell（100μl）Melt in ice bath.10 μ l are added
Liquid is connected, gently mixing is placed on ice bath 30 minutes.42 DEG C, after 30 seconds heat shocks, ice bath cools down 2 minutes.Add SOC culture mediums
900 μ L are cultivated 1 hour in 37 DEG C of vibrations.It takes and is applied on LB plates in right amount, be incubated overnight in 37 DEG C.
10, the determination of recombinant：Doubtful positive single bacterium colony uniform from picking size on LB agar plates, form is mellow and full,
Each bacterium colony to be checked chosen by sterile working sterile to 1.5 mL of the LB liquid medium containing 30 μ L ampicillins
In centrifuge tube, pipettor is blown and beaten uniformly at bacterial suspension, while being done template with this suspension and being carried out bacterium colony PCR identifications, primer
It is as described in Table 1 to sequence.Each section of gene magnification carries out amplification identification with corresponding primer respectively, and system is shown in Table 4.
Table 4 is colony identification PCR system：
| 2xTaq PCR Master Mix | 10.0μL |
| P1(10μM) | 0.5μL |
| P2(10μM) | 0.5μL |
| Bacterium colony template | 1.0μL |
| ddH2O | 8.0μL |
| Total | 20.0μL |
PCR amplification program：
Amplified production chooses the corresponding bacterium solution that can amplify strong and special purpose band to containing ammonia into row agarose gel electrophoresis
Bacterium is shaken in the liquid medium of parasiticin, bacterium solution Song Jinwei intelligence Bioisystech Co., Ltd is sequenced.
11, the processing of gene sequencing result
Sequencing result is removed using the Editseq softwares in 7.10 bioinformatics softwares of Lasergene including primer pair
Export-oriented terminal sequence inside, the splicing of full length sequence and homology analysis quickly compare tool simultaneously, with GenBank in NCBI
BLAST carries out the verification of sequencing result or compares search, completes sequence and is compared with the existing sequence in database.Stream
Row strain cell passes on the 65th generation virus O RF3 genes complete sequence such as SEQ ID NO：Shown in 17.
Sequence table
<110>Agricultural University Of South China
<120>A kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 1
atggctagca accaagtcac attggcttt 29
<210> 2
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 2
ggtttaaatg tcgtctcttc taattccac 29
<210> 3
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 3
gagaagttta aagtcttcct taactgtgt 29
<210> 4
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 4
caacaacagg tttgttgtct gtgacaacg 29
<210> 5
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 5
cacgtagtgt taaggctgtt aaagtagaa 29
<210> 6
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 6
atgaaggtta tacccttaac tttagtcgt 29
<210> 7
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 7
ggaagaaact aggaagtaca ttatccgta 29
<210> 8
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 8
ctaattctga ccaaaagaaa gtcagaaaa 29
<210> 9
<211> 26
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 9
gcgcctgcag aaatgtcttg ctggtt 26
<210> 10
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 10
cgaacacagt tcaccttgag gaatttacc 29
<210> 11
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 11
ctacaacaag gatgttgctt gtctaggta 29
<210> 12
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 12
cagatgctta acccaatcca aaagcacat 29
<210> 13
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 13
gaaaaccctg aatataagaa ggtgtttta 29
<210> 14
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 14
tgtcaactcc aaaccaccag cccacaaca 29
<210> 15
<211> 29
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 15
ctacctggcc aacctatcag acatactaa 29
<210> 16
<211> 33
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 16
tcatttgttt acgttgacca aatgattaga aaa 33
<210> 17
<211> 20343
<212> DNA
<213>Porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
<400> 17
atggctagca accaagtcac attggctttt gccaatgatg cagaaatttc agcttttggc 60
ttttgcactg ctagtgaagc cgtctcatac tattctgagg ccgccgctag tgggtttatg 120
caatgccgtt tcgtgtcctt cgatctcgct gacactgttg agggattgct tcccgaagac 180
tatgtcatgg tggtggtcgg cactaccaag cttagtgcgt atgtggacac ttttggtagc 240
cgccccagaa acatttgtgg ttggctgtta ttttctaact gtaattactt cctcgaagag 300
ttagagctca cttttggtcg tcgtggtggt aacatcgtgc cagttgacca atacatgtgt 360
ggcgctgacg ggaaacctgt tcttcaggaa tccgagtgga gtatacagac ttttttgctg 420
actccgaaga cggtcaactc aacattgctg ggatcactta tgtgaaggcc tggattgtag 480
agcgatcgga cgtctcttat gcgagtcaga atttaacatc tattaagtct attacttact 540
gttcaaccta tgagcatact tttcctgatg gtactgccat gaaggttgca cgtactccaa 600
agattaagaa gactgttgtc ttgtctgagc cacttgctac tatctacagg gaaattggtt 660
ctccttttgt ggataatggg agcgatgctc gttctatcat taagagacca gtgttcctcc 720
acgcttttgt taagtgtaag tgtggtagtt atcattggac tgttggtgat tggacttcct 780
atgtctccac ttgctgtggc tttaagtgta agccagtcct tgtggcttca tgctctgcta 840
cgcctggttc tgttgtggtt acgcgcgctg gtgctggcac tggtgttaag tattacaaca 900
acatgttcct gcgccatgtg gcagacattg atgggttggc attctggcga attcttaagg 960
tgcagtccaa agacgatctc gcttgctctg gtaaattcct tgaacaccat gaggaaggtt 1020
tcacagatcc ttgctacttt ttgaatgact cgagcattgc tactaagctc aagtttgaca 1080
tccttagtgg caagttttct gatgaagtca aacaagctat ctttgctggt catgttgttg 1140
ttggcagtgc gctcgttgac attgttgacg atgcactggg acagccttgg tttatacgta 1200
agcttggtga ccttgcaagt gcagcttggg agcagcttaa ggctgtcgtt agaggcctta 1260
acctcctgtc tgatgaggtc gtgctctttg gcaaaagact tagctgtgcc actcttagta 1320
tcgttaacgg tgtttttgag tttatcgccg aagtgccaga gaagttggct gcggctgtta 1380
cagtttttgt caacttcttg aatgagcttt ttgagtctgc ctgtgactgc ttaaaggtcg 1440
gaggtaaaac ctttaacaag gttggctcct atgttctttt tgacaacgca ttggttaagc 1500
ttgtcaaggc aaaagttcgc ggcccacgac aggcaggtgt ttgtgaagtt cgttacacaa 1560
gccttgttat tgggagtact accaaggtgg tttccaagcg cgttgaaaat gccaatgtga 1620
atctcgtcgt tgttgacgag gatgtgaccc tcaacaccac tggtcgtaca gttgttgttg 1680
acggacttgc attcttcgag agtgacgggt tttacagaca tcttgctgat gctgacgttg 1740
tcattgaaca tcctgtttat aagtctgctt gtgagctcaa gccagttttt gagtgtgacc 1800
caatacctga ttttcctatg cctgtggccg ctagtgttgc agagctttgt gtgcaaactg 1860
atctgttgct taaaaattac aacactcctt ataaaactta cagctgcgtt gtgagaggtg 1920
ataagtgttg catcacttgc accttacata tcacagcacc aagttatatg gaggatgctg 1980
ctaattttgt agacctctgt accaggaaca ttggtactgc tggttttcat gagttttaca 2040
ttacggccca tgaacaacag gatctgcaag ggttcgtaac cacttgttgc acgatgtcag 2100
gttttgagtg ttttatgcct ataatcccac agtgtccagc agtgcttgaa gagattgatg 2160
gtggtagcat ctggcggtct tttatcactg gtcttaatac aatgtgggat ttttgcaagc 2220
atcttaaagt cagctttgga ctagatggca ttgttgtcac tgtagcacgc aaatttaaac 2280
gacttggtgc tctcttggca gaaatgtata acacttacct ttcaactgtg gtggaaaact 2340
tggtactggc cggtgttagc ttcaagtatt atgccaccag tgtcccaaaa attgttttgg 2400
gctgttgttt tcacagtgtt aaaagtgttc ttgtaagtgc cttccagatt cctgtccagg 2460
caggcattga gaagtttaaa gtcttcctta actgtgttca ccctgttgta ccacgcgtca 2520
tcgaaacttc ttttgtggaa ttagaagaga cgacatttaa accaccagca ctcaatggta 2580
gtattgctat tgttgatggt tttgctttct attatgatgg aacactatac tatcccaccg 2640
atggtaatag tgttgtgcct atctgtttta agaagaaggg tggtggtgat gtcaaattct 2700
ctgatgaagt ctctgttaga accattgacc cagtttataa ggtctccctt gaatttgagt 2760
tcgagtctga gactattatg gctgtgctta ataaggctgt tggtaatcgt atcaaggtta 2820
caggtggttg ggacgatgtt gttgagtata tcaacgttgc cattgaggtt cttaaagatc 2880
atatcgatgt gcctaagtac tacatctatg atgaggaagg tggcaccgat cctaatctga 2940
ccgtaatggt ttctcagtgg ccgttgaatg atgacacgat ctcacaggat ctgcttgatg 3000
tggaaattgt tactgatgca ccaattgatt tcgagggtga tgaagtagac tcctctgacc 3060
ctgataaggt ggcagatgtg gctaactctg agcctgagga tgatggtcct aatgtagctc 3120
ctgaaacaaa tgtagagtct gaagttgagg aagttgccgc aaccttgtcc tttattaaag 3180
atacaccttc cacagttact aaggatcctt ttgcttttga ctttgtaagc tatggaggac 3240
ttaaggtttt aagacaatct cataacaact gctgggttac ttctaccttg gtgcagctac 3300
aattgcttgg catcgttgat gaccctgcaa tggagctttt tagtgctggt agagttggtc 3360
caatggttcg caaatgctat gagtcacaaa aggctatctt gggatctttg ggtgatgtgt 3420
cggcttgcct agagtctctg actaaggacc tacacacact taagattacc tgttctgtag 3480
tctgtggttg tggtactggt gaacgtatct atgagggttg tgcttttcgt atgacgccaa 3540
ctttggaacc gttcccatat ggtgcttgtg ctcagtgtgc tcaagttttg atgcacactt 3600
ttaaaagtat tgttggcacc ggcatctttt gtcgagatac tactgctctc tccttggatt 3660
ctttggttgt aaaacctctt tgtgcggctg cttttatagg caaggatagt ggtcattatg 3720
tcactaactt ttatgatgct gctatggcta ttgatggtta tggtcgtcat cagataaagt 3780
atgacacact gaacactatt tgtgttaaag acgttaattg gacagcacct tttgtcccag 3840
acgttgagcc tgtattggag cctgttgtca aaccgttcta ttcttataag aatgttgatt 3900
tttaccaagg agattttagt gaccttgtta aacttccatg tgactttgtt gttaatgctg 3960
caaatgagaa tttgtctcac ggtggcggca tagcaaaggc cattgatgtt tataccaagg 4020
gcatgctgca gaagtgctca aatgattaca ttaaagcaca cggttccatt aaagttggac 4080
gtggtgtcat gttggaggca ttaggtctta aggtctttaa tgttgttggt ccacgtaagg 4140
gtaagcatgc acctgagctt cttgttaagg cttataagtc cgtttttgct aattcaggtg 4200
ttgctcttac acctttgatt agtgttggaa tttttagtgt tcctttggaa gaatctttat 4260
ctgcttttct tgcatgtgtt ggtgatcgcc actgtaagtg cttttgttat agtgacaaag 4320
agcgcgaggc gatcattaat tacatggatg gcttggtaga tgctattttc aaagatgcgc 4380
ttgttgatac tactcccgtc caggaagatg ttcaacaagt ttcacaaaaa ccagttttgc 4440
ctaattttga acctttcagg attgaaggtg ctcatgcttt ctatgagtgt aaccctgaag 4500
gtttgatgtc cttaggtgct gacaagctgg tgttgtttac aaattccact ttggattttt 4560
gtagcgttgg taagtgtctt aacaatgtga ccggcggtgc attgcttgaa gccataaatg 4620
tatttaaaaa gagtaacaaa acagtgcctg ctggcaactg tgttactttt gagtgtgcag 4680
atatgatttc tattactatg gtagtattgc cagctgatgg tgatgctaat tatgacaaaa 4740
attatgcacg cgccgtcgtc aaggtatcta agcttaaagg caagttattg cttgttgttg 4800
gtgatgccac gttgtattcc aagttgtccc acctcagcgt gataggtttc gtatccacac 4860
ctgatgatgt ggagcgtttc tacgcaaata agagtgtggt tattaaagtc actgaggata 4920
cacgtagtgt taaggctgtt aaagtagaat ccactgttac ttatggacaa caaattggac 4980
cttgtcttgt taatgacacc gttgtcacag acaacaaacc tgttgttgct gacgttgtag 5040
ctaaggttgt accaagtgct aattgggatt cacattatgg ttttgataag gctggtgagt 5100
tccacatgct agaccatact gggtttgcct ttcctagtga agttgttaac ggtaggcgtg 5160
tgcttaaaac cacagataat aactgttggg ttaatgttac atgtttacaa ttacagtttg 5220
ctagatttag gttccagtca gcaggtctac aggctatgtg ggagtcctat tgtactggtg 5280
atgttgctat gtttgtgcat tggttgtact ggcttactgg tgttgacaaa ggtcagccta 5340
gtgattcaga aaatgcactt aacatgttgt ccaagtacat tgtttctgct ggttctgtca 5400
ctattgaacg tgtcacgcat gacggctgtt gttgtagtaa gcgtgttgtc actgcaccag 5460
ttgtgaatgc tagcgtattg aagcttggcg tcgaggatgg tctttgtcca catggtctta 5520
actacattga caaagttgtt gtagttaaag gtactacaat tgttgtcaat gttggaaaac 5580
ctgtagtggc accatcacac ctctttctta agggtgtttc ctacacaaca ttcctagata 5640
atggtaacgg tgttgtcggc cattatactg tttttgatca tgacactggt atggtgcatg 5700
atggagatgc ttttgtaccg ggtgatctca atgtatctcc tgttacaaat gttgtcgtct 5760
cagagcagac tgctgttgtg attaaagacc ctgtgaagaa agtagagtta gacgctacaa 5820
agctgttaga cactatgaat tatgcatcgg aaagattctt ttcctttggt gattttatgt 5880
cacgtaattt aattacagtg tttttgtaca tccttagcat tttgggtctc tgttttaggg 5940
cctttcgtaa gagggatgtt aaagttctag ctggtgtacc ccaacgtact ggtattatat 6000
tgcgtaaaag tgtgcgctat aatgcaaagg cgttgggtgt cttcttcaag ctaaaacttt 6060
attggttcaa agttcttggt aagtttagtt tgggtattta tgcattgtat gcattactat 6120
tcatgacaat acgctttaca cctataggtg gccctgtttg tgatgatgtt gttgctggtt 6180
atgctaattc tagttttgac aagaatgagt attgcaacag tgttatttgt aaggtctgtc 6240
tctatgggta ccaggaactt tcggactttt ctcacacaca ggtagtatgg caacacctta 6300
gagacccgtt aattggtaat gtgatgcctt tcttttattt ggcatttctg gcaatttttg 6360
ggggtgttta tgtaaaggct attactctct attttatttt ccagtacctt aacattcttg 6420
gtgtgttttt gggcctacaa cagtccattt ggtttttgca gcttgtgcct tttgatgtct 6480
ttggtgacga gatcgtcgtc tttttcatcg ttacacgcgt attgatgttc cttaagcatg 6540
ttttccttgg ctgcgataag gcatcttgtg tggcttgctc taagagtgca cgccttaagc 6600
gcgttcctgt ccagactatt tttcagggta ctagcaaatc cttctacgta catgccaatg 6660
gtggttctaa gttctgtaag aagcacaatt tcttttgttt aaattgtgat tcttatggtc 6720
caggctgcac ttttattaat gacgtcattg caactgaagt tggtaatgtt gtcaaactta 6780
atgtgcaacc gacaggtcct gccactattc ttatcgacaa ggttgaattc agtaatggtt 6840
tttactatct ttatagtggt gacacatttt ggaagtacaa ctttgacata acagatagca 6900
aatacacttg caaagaatca cttaaaaatt gtagcataat cacagacttt attgttttta 6960
acaataatgg ttccaatgta aatcaggtta agaatgcatg tgtttatttt tcacagatgc 7020
tttgtaaacc tgttaagtta gtggactcag cgttgttggc cagtttgtct gttgattttg 7080
gtgcaagctt acatagtgct tttgttagtg tgttgtcgaa tagttttggc aaagatctgt 7140
caagttgtaa tgacatgcag gattgcaaga gcacattggg ttttgatgat gtaccattgg 7200
acacctttaa tgttgctgtt gctgaggctc atcgttacga tgtcctcttg actgacatgt 7260
cgttcaacaa ttttaccacc agttatgcaa aaccagagga aaaacttcct gtccatgaca 7320
ttgccacgtg tatgcgtgta ggtgccaaga ttgttaatca taacgttctt gtcaaggata 7380
gtatacctgt ggtgtggctt gtacgtgatt tcattgccct ttcggaagaa actaggaagt 7440
acattatccg tacgactaaa gttaagggta taaccttcat gttgaccttt aatgattgtc 7500
gtatgcatac taccatacct actgtttgca ttgcaaataa gaagggtgca ggtcttccta 7560
gtttttcaaa ggttaagaaa ttcttctggt ttttgtgtct gttcatagtt gttgttttct 7620
ttgcactaag ctttcttgat tttagtactc aggttagcag tgatagtgat tatgacttca 7680
agtatattga gagtggccag ttgaagactt ttgacaatcc acttagttgt gtgcataatg 7740
tctttagtaa cttcgaccag tggcatgatg ccaagtttgg tttcaccccc gtcaacaatc 7800
ctagttgtcc tatagtcgtt ggtgtatcag acgaagctcg cactgttcca ggtatcccag 7860
caggtgttta tttagctggt aaaacacttg tttttgctat taacaccatt tttggtacat 7920
ctggtttgtg ctttgatgct agtggcgttg ctgataaggg cgcttgcatt tttaattcgg 7980
cttgcaccac attatctggt ttgggtggaa ctgctgtcta ctgttataag aatggtctag 8040
ttgaaggtgc taaactttat agtgagttgg cacctcatag ctactataaa atggtagatg 8100
gtaatgctgt gtctttacct gaaattatct cacgcggctt tggcatccgt actatccgta 8160
caaaggctat gacctactgt cgcgttggcc agtgtgtgca atctgcagaa ggtgtttgtt 8220
ttggcgccga tagattcttt gtctataatg cagaatctgg ttctgacttt gtttgtggca 8280
caggtctctt tacattgttg atgaacgtta ttagtgtttt ttccaagaca gtaccagtaa 8340
ctgtgttgtc tggtcaaata ctttttaatt gcattattgc ttttgctgct gttgcggtgt 8400
gtttcttatt tacaaagttt aagcgcatgt tcggtgatat gtctgttggc gttttcactg 8460
tcggtgcttg tactttgttg aacaatgttt cctacattgt aacacagaac acacttggca 8520
tgttgggcta tgcaactttg tactttttgt gcactaaagg tgttagatat atgtggattt 8580
ggcatttggg atttttgatc tcatatatac ttattgcacc atggtgggtt ttgatggttt 8640
atgccttttc agccattttt gagtttatgc ctaacctttt taagcttaag gtttcaacac 8700
aactttttga gggtgacaag ttcgtaggct cttttgaaaa tgctgcagca ggtacatttg 8760
tgcttgacat gcatgcctat gagagacttg ccaactctat ctcaactgaa aaactgcgtc 8820
agtatgctag tacctacaat aagtacaagt attattcagg cagtgcttca gaggctgatt 8880
acaggcttgc ttgttttgcc catttggcca aggctatgat ggattatgct tctaagcaca 8940
acgacacgtt atacacacca cccactgtga gttacaattc aactctacag gctggcttgc 9000
gtaagatggc acaaccatct ggtgttgttg agaagtgcat agttcgtgtt tgctatggta 9060
atatggctct taatggccta tggcttggtg atactgttat gtgcccacgc catgttatag 9120
cgtctagtac tactagcact atagattatg actatgccct ttctgtttta cgcctccaca 9180
acttctccat ttcatctggt aatgttttcc taggtgttgt gggtgtaacc atgcgaggtg 9240
ctttgttgca gataaaggtt aatcaaaaca atgtccacac gcctaagtac acctatcgca 9300
cagttagacc gggtgaatct tttaatatct tggcgtgcta tgatggtgct gcagctggtg 9360
tttacggcgt taacatgcgc tctaattaca ctattagagg ctcgttcatt aatggcgctt 9420
gtggttcacc tggttacaac attaacaatg gtaccgttga gttttgctat ttacaccagc 9480
ttgaacttgg ttcaggccgt catgttggta gcgacttaga tggtgttatg tatggtggtt 9540
atgaggacca acctactttg caagttgaag gcgctagtag tctgtttaca gagaatgtgt 9600
tggcatttct ttatgcagca ctcattaatg gttctacctg gtggcttagt tcttctagga 9660
ttgctgtaga caggtttaat gagtgggctg ttcataatgg tatgacaaca gtagttaata 9720
ctgattgctt ttctattctt gctgctaaga ctggtgttga tgtacaacgt ttgttggcct 9780
caatccagtc tctgcataag aattttggtg gaaagcaaat tcttggctat acctcgttga 9840
cagatgagtt tactacaggt gaagttatac gtcaaatgta tggcgttaat cttcagagtg 9900
gttatgtttc acgcgcctgc agaaatgtct tgctggttgg ttcttttctg actttctttt 9960
ggtcagaatt agtttcttac actaagttct tttgggtaaa tcctggttat gtcacaccta 10020
tgtttgcgtg tttgtcattg ctgtcctcac ttttgatgtt cacactcaag cataagacat 10080
tgtttttcca ggtctttcta atacctgctc tgattgttac atcttgcatt aatttggcat 10140
ttgatgttga agtctacaac tatttggcag agcattttga ttaccatgtt tctctcatgg 10200
gttttaatgc acaaggtctt gttaacatct ttgtctgctt tgttgttacc attttacacg 10260
gcacatacac atggcgcttt tttaacacac ctgtgagttc tgtcacttat gtggtagctt 10320
tgctgactgc ggcatataac tatttttacg ctagtgacat tcttagttgt gctatgacac 10380
tatttgctag tgtgactggc aactggttcg ttggtgctgt ttgttataaa gctgctgttt 10440
atatggcctt gagatttcct acttttgtgg ctatttttgg tgatattaag agtgttatgt 10500
tctgttacct tgtgttgggt tattttacct gttgcttcta tggtattctc tactggttca 10560
acaggttctt taaggttagt gtaggtgtct atgactatac tgttagtgct gctgagttta 10620
agtatatggt tgctaacggc ctacgtgcac caactggaac acttgattca ctacttctgt 10680
ctgccaaatt gattggtatt ggtggtgagc ggaatattaa gatttcttcc gttcagtcta 10740
aactgactga tattaagtgt agtaacgttg tgcttttagg ctgtctctct agcatgaatg 10800
tctcagcaaa ttcaacagaa tgggcctatt gtgttgactt gcataacaag atcaacttgt 10860
gtaatgaccc agaaaaagcg caggaaatgc tacttgcttt gttggcattt ttccttagta 10920
agaatagtgc ttttggttta gatgacttat tggaatccta ttttaatgac aatagtatgt 10980
tgcagagtgt tgcatctact tatgtcggtt tgccttctta tgtcatttat gaaaatgcac 11040
gccaacagta tgaagatgct gttaataatg gttctccacc tcagttggtt aagcaattgc 11100
gccatgctat gaatgtggca aagagtgaat ttgaccgtga ggcttctact cagcgtaagc 11160
ttgatagaat ggctgagcag gctgcagcac agatgtataa agaggcaaga gcagttaata 11220
ggaagtctaa agttgtaagt gctatgcatt cactgctttt tggtatgttg agacgtttgg 11280
atatgtcttc tgtagacact attctcaact tggcaaagga tggggttgta cctctgtctg 11340
tcataccggc agtcagtgct actaagctta acattgttac ttctgatatc gattcttata 11400
atcgtatcca gcgtgaggga tgtgtccact acgctggtac catttggaat ataattgata 11460
tcaaggacaa tgatggcaag gtggtacacg ttaaggaggt aaccgcacag aatgctgagt 11520
ccctgtcatg gcccctggtc cttgggtgtg agcgtattgt caagctccag aataatgaaa 11580
ttattcctgg taagctgaag cagcgctcca ttaaggcaga aggagatggc atagttggag 11640
aaggtaaggc actttacaat aatgagggtg gacgtacttt tatgtatgct ttcatctcgg 11700
acaaaccgga cctgcgtgta gttaagtggg agttcgatgg tggttgtaac actattgagc 11760
tagaaccacc acgtaagttc ttggtggatt ctcctaatgg tgcacagatc aagtatctct 11820
actttgttcg taaccttaac acgttacgta ggggtgctgt tctcggctac ataggtgcca 11880
ctgtacgctt gcaggctggt aaacaaacag aacaggctat taactcttca ttgttgacac 11940
tttgcgcttt cgctgtggat cctgctaaga cctacatcga tgctgtcaaa agtggtcaca 12000
aaccagtagg taactgtgtt aagatgttgg ccaatggttc tggtaatgga caagctgtta 12060
ctaatggtgt ggaggctagt actaaccagg attcatatgg tggtgcgtcc gtgtgtctat 12120
attgtagagc acatgttgag catccatcta tggatggttt ttgcagactg aaaggcaagt 12180
acgtacaggt tccactaggt acagtggatc ctatacgttt tgtacttgag aatgacgttt 12240
gcaaggtttg tggttgttgg ctggctaatg gctgcacttg tgacagatcc attatgcaaa 12300
gtactgatat ggcttattta aacgagtacg gggctctagt gcagctcgac tagagccctg 12360
taacggtact gatacacaac atgtgtatcg tgcttttgac atctacaaca aggatgttgc 12420
ttgtctaggt aaattcctca aggtgaactg tgttcgcctg aagaatttgg ataagcatga 12480
tgcattctat gttgtcaaaa gatgtaccaa gtctgcgatg gaacacgagc aatccatcta 12540
tagcagactt gaaaagtgtg gagccgtagc cgaacacgat ttcttcactt ggaaggatgg 12600
tcgtgctatc tatggtaacg tttgtagaaa ggatcttacc gagtatacta tgatggattt 12660
gtgttacgct ttacgtaact ttgatgaaaa caattgtgat gttcttaaga gcattttaat 12720
taaggtaggc gcttgtgagg agtcctactt caataataaa gtctggtttg accctgttga 12780
aaatgaagac attcatcgtg tctatgcatt gttaggtacc attgtttcac gtgctatgct 12840
taaatgcgtt aagttctgtg atgcaatggt tgaacaaggt atagttggtg ttgtcacatt 12900
agataatcag gatcttaatg gtgattttta tgattttggt gattttactt gtagcatcaa 12960
gggaatgggt atacccattt gcacatcata ttactcttat atgatgcctg ttatgggtat 13020
gactaattgc cttgctagtg agtgttttgt taagagtgat atatttggtg aggatttcaa 13080
gtcatatgac ctgctggaat atgatttcac ggagcataag acagcactct tcaacaagta 13140
tttcaagtat tggggactgc aataccaccc taactgtgtg gactgcagtg atgagcagtg 13200
catagttcac tgtgccaact tcaatacgtt gttttccact actataccta ttacggcatt 13260
tggacctttg tgtcgcaagt gttggattga tggtgttcca ctggtaacta cagctggtta 13320
tcattttaaa cagttaggta tagtttggaa caatgacctc aacttacact ctagcaggct 13380
ctctattaac gaactactcc agttttgtag tgatcctgca ttgcttatag catcatcacc 13440
agcccttgtt gatcagcgta ctgtttgctt ttcagttgca gcgctaggta caggtatgac 13500
taaccagact gttaaacctg gccatttcaa taaggagttt tatgacttct tacttgagca 13560
aggtttcttt tctgagggct ctgagcttac tttaaagcac ttcttctttg cacagaaggg 13620
tgatgcagct gttaaggatt ttgactacta taggtataat agacctactg ttctggacat 13680
ttgccaagct cgcgtcgtgt atcaaatagt gcaacgctat tttgatattt acgaaggtgg 13740
ttgtatcact gctaaagaag tggttgttac aaaccttaac aagagcgcag gttatccttt 13800
gaacaagttt ggtaaagctg gtctttacta tgagtcttta tcctatgagg aacaggatga 13860
actttatgct tatactaagc gtaacatcct gcccactatg acacagctca accttaaata 13920
tgctataagt ggcaaagaac gtgcacgcac agtgggtggt gtttcgcttt tgtcaaccat 13980
gactactcgg cagtatcatc aaaaacacct taagtccata gttaatacta ggggcgcttc 14040
ggttgttatt ggtactacta agttttatgg tggttgggac aatatgctta agaaccttat 14100
tgatggtgtt gaaaatccgt gtcttatggg ttgggactac ccaaagtgcg acagagcact 14160
gcccaatatg atacgcatga tttcagccat gattttaggc tctaagcaca ccacatgctg 14220
cagttccact gaccgctttt tcaggttgtg caatgaattg gctcaagtcc ttactgaggt 14280
tgtttattct aatggaggtt tttatttgaa gccaggtggt actacctctg gtgatgcaac 14340
caccgcatat gcaaactcag ttttcaatat cttccaagca gtaagtgcca atgttaacaa 14400
acttcttagt gttgacagca atgtctgtca taatttagaa gttaagcaat tgcagcgtaa 14460
gctttatgag tgctgttata gatcaactac cgtcgatgac cagttcgtcg ttgagtatta 14520
tggttacttg cgtaaacatt tttcaatgat gattctttct gatgatggcg ttgtttgtta 14580
taacaatgac tatgcatcac ttggttatgt tgctgatctt aacgcattca aggctgtttt 14640
gtattaccag aacaatgtct tcatgagcgc ctctaaatgt tggatcgagc ctgacattaa 14700
taaaggtcct catgaatttt gctcgcagca tactatgcag attgtcgata aagatggtac 14760
ttattacctt ccttaccctg atccttcaag aatcctctct gcaggtgtgt ttgttgatga 14820
cgttgttaaa actgatgcag ttgtattgct tgaacgttat gtgtcattgg ctatagatgc 14880
ctacccgtta tctaagcatg aaaaccctga atataagaag gtgttttatg tgcttttgga 14940
ttgggttaag catctgtaca aaactttgaa tgctggtgtg ttagagtctt tttctgtcac 15000
acttttggaa gattctactg ctaaattctg ggatgagagc ttttatgcca acatgtatga 15060
gaaatctgca gttttacaat ctgcagggct ttgtgttgtt tgtggctctc aaactgtttt 15120
acgttgtggt gattgtctac ggcgtcctat gctttgtact aagtgtgctt atgatcatgt 15180
cattggaaca actcacaagt tcattttggc catcactcca tatgtgtgtt gtgcttcaga 15240
ttgtggtgtc aatgatgtaa ctaagctcta cttaggtggt cttagttatt ggtgtcatga 15300
acacaagcca cgtcttgcat tcccgttgtg ctctgctggt aatgtttttg gcttgtacaa 15360
aaattctgct accggctcac ccgatgttga agactttaat cgcattgcta catccgattg 15420
gactgatgtt tctgactaca ggttggcaaa tgatgtcaag gactcattgc gtctgtttgc 15480
agcggaaact atcaaggcca aggaggagag cgttaagtca tcctacgctt gtgcaacact 15540
acatgaggtt gtaggaccta aagagttgtt gctcaaatgg gaagtcggca gacccaaacc 15600
accccttaat agaaattcgg ttttcacttg ttatcatata acgaagaaca ccaaatttca 15660
aatcggtgag tttgtgtttg agaaggcaga atatgataat gatgctgtaa catataaaac 15720
taccgccaca acaaaacttg ttcctggcat ggtttttgtg cttacctcac ataatgttca 15780
gccattgcgc gcaccgacca ttgctaatca agaacgttat tccactatac ataagttgca 15840
tcctgctttt aacatacctg aagcttattc tagcttagtg ccctattacc aactgattgg 15900
taagcagaag attacaacta tccagggacc tcccggtagt ggtaaatctc actgtgttat 15960
agggctaggt ttgtactatc caggtgcacg tatagtgttt acagcttgtt ctcatgcagc 16020
ggtcgattca ctttgtgtga aagcctccac tgcttatagc aatgacaaat gttcacgcat 16080
cataccacag cgtgctcgtg ttgagtgtta tgacggtttc aagtctaata atactagtgc 16140
tcagtacctt ttctccactg tcaatgcttt gccagagtgc aatgcggaca ttgttgtggt 16200
ggatgaggtc tctatgtgca ctaattatga cttgtctgtc ataaatcagc gcatcagcta 16260
taggcatgta gtctatgttg gtgaccctca acagctgcct gcaccacgtg ttatgatttc 16320
acgtggtact ttggaaccaa aggactacaa cgttgtcact caacgcatgt gtgcccttaa 16380
gcctgatgtt ttcttgcaca agtgttatcg ctgtcctgct gagatagtgc gtactgtgtc 16440
tgagatggtc tatgaaaacc aattcattcc tgtgcaccca gatagcaagc agtgttttaa 16500
aatcttttgc aagggtaatg ttcaggttga caatggttca agtattaatc gcaggcaatt 16560
ggatgttgtg cgtatgtttt tggctaaaaa tccgaggtgg tcaaaggctg tttttatttc 16620
tccttataac agccagaatt atgttgccag ccgcatgcta ggtttacaaa ttcagacagt 16680
tgactcatcc cagggtagtg agtatgacta tgtcatttac acacaaactt cagatactgc 16740
ccatgcctgt aatgttaaca ggtttaatgt tgccatcaca agagctaaga aaggcatatt 16800
atgtataatg tgcgataggt ccctttttga tgtgcttaaa ttctttgagc ttaaattgtc 16860
tgatttgcag gctaatgggg gttgtggtct ttttaaagac tgtagcagag gtgatgatct 16920
gttgccacca tctcacgcta acaccttcat gtctttagcg gacaatttta agactgatca 16980
agatcttgct gttcaaatag gtgttaatgg acccattaaa tatgagcatg ttatctcgtt 17040
tatgggtttc cgttttgata tcaacatacc caaccatcat actctctttt gcacacgcga 17100
ctttgccatg cgtaatgtta gaggttggtt aggttttgac gttgaaggag cacatgttgt 17160
tggctctaac gtcggtacaa atgtcccatt gcaattaggg ttttctaacg gtgttgattt 17220
tgttgtcaga cctgaaggtt gcgttgtaac agagtctggt gactacatta aacccgtcag 17280
agctcgtgct ccaccagggg aacaattcgc acaccttttg cctctactta aacgcggcca 17340
accatgggat gttgtccgca aacgtatagt gcagatgtgt agtgactacc tggccaacct 17400
atcagacata ctaatttttg tgttgtgggc tggtggtttg gagttgacaa ctatgcgtta 17460
ttttgtcaag attggaccaa gtaagagttg tgattgtggt aaggttgcta cttgttacaa 17520
tagtgcgctg catacgtact gttgtttcaa acatgccctt ggttgtgatt atctgtataa 17580
cccatactgt attgatatac agcagtgggg atacaaggga tcacttagcc ttaaccacca 17640
tgagcattgt aatgtacata gaaacgagca tgtggcttct ggtgatgcca taatgactcg 17700
ctgtctggcc atacatgatt gctttgtcaa gaacgttgac tggtccatca catacccatt 17760
tattggtaat gaggctgtta ttaataagag cggccgaatt gtgcaatcac acaccatgcg 17820
gtcagttctt aagttataca atccgaaagc catatatgat attggcaatc ctaagggcat 17880
cagatgtgcc gtaacggatg ctaagtggtt ttgctttgac aagaatccta ctaattctaa 17940
tgtcaagaca ttggagtatg actatataac acatggccaa tttgatgggt tgtgcttgtt 18000
ttggaattgc aatgtagaca tgtatccaga attttctgtg gtctgtcgtt ttgtactcgc 18060
tgtaggtcac cactcaactt ggagggttgt aatggtggtt cactgtatgt taataatcat 18120
gcattccata caccggcttt tgacaagcgt gcttttgcta agttgaagcc aatgccattt 18180
ttcttttatg atgatactga gtgtgacaag ttacaggact ccataaacta tgttcctctt 18240
agggctagta actgcattac taaatgtaat gttggtggag ctgtctgtag taagcattgt 18300
gctatgtatc atagctatgt taatgcttac aacactttta cgtcggcggg ctttactatt 18360
tgggtgccta cttcgtttga cacctataat ctgtggcaga catttagtaa caatttgcaa 18420
ggtcttgaga acattgcttt caatgtcgta aagaaaggat cttttgttgg tgccgaaggt 18480
gaacttcctg tagctgtggt taatgacaaa gtgctcgtta gagatggtac tgttgatact 18540
cttgttttta caaacaagac atcactaccc actaacgtag cttttgagtt gtatgccaag 18600
cgtaaggtag gactcacccc acccattacg atcctacgta acttgggtgt agtttgtaca 18660
tctaagtgtg tcatttggga ctatgaagcc gaacgtccac ttactacttt tacaaaggat 18720
gtttgtaaat ataccgactt tgagggtgac gtctgtacac tctttgataa cagcattgtt 18780
ggttcattag agcgattctc tatgacccaa aatgctgtgc ttatgtcact tacagccgtt 18840
aaaaagctta ctggcataaa gttaacttat ggttatctta atggtgtccc agttaacaca 18900
catgaagata aaccttttac ttggtatatt tacactagga agaacggcaa attcgaggac 18960
tatcctgatg gctattttac ccaaggtaga acaaccgctg attttagccc tcgtagcgat 19020
atggaaaagg acttcctaag tatggatatg ggtctgttta ttaacaagta cggacttgaa 19080
gattacggct ttgagcacgt tgtgtatggt gatgtttcaa aaaccaccct tggtggttta 19140
catctactaa tttcgcaggt gcgtctgtcc tgtatgggtg tgcttaaaat agacgagttt 19200
gtgtctagta atgatagcac gttaaagtcc tgtactgtta catatgctga taaccctagt 19260
agtaagatgg tttgcacgta tatggatctc ctgcttgacg attttgtcag cattcttaaa 19320
tcgttggatt tgagcgttgt atctaaagtt catgaagtta tggtcgattg taaaatgtgg 19380
aggtggatgt tgtggtgtaa ggatcataaa ctccagacat tttatccgca acttcaggcc 19440
agtgaatgga agtgtggtta ttccatgcct tctatttaca agatacaacg tatgtgttta 19500
gaaccttgca atctctataa ctatggcgct ggtattaagt tacctgatgg cattatgttt 19560
aacgtagtta aatacacaca gctttgtcaa tatctcaata gcaccacaat gtgtgtaccc 19620
catcacatgc gtgtgctaca tcttggtgct ggctccgata agggtgttgc acctggcacg 19680
gctgtcttac gacgttggtt gccactggat gccattatag ttgacaatga tagtgtggat 19740
tacgttagcg atgctgatta tagtgttaca ggagattgct ctaccttata cctgtcagat 19800
aagtttgact tagttatatc tgatatgtat gacggtaaga ttaaaagttg tgatggggag 19860
aacgtgtcta aagaaggctt ctttccctat attaatggtg tcatcactga aaagttggca 19920
cttggtggta ctgtggctat taaggtgacg gagtttagtt ggaataagaa gttgtatgaa 19980
ctcattcaga agtttgagta ttggacaatg ttctgtacca gtgttaacac gtcatcgtca 20040
gaggcatttt taattggtgt tcactattta ggtgattttg caagtggcgc tgttattgac 20100
ggcaacacta tgcatgccaa ttatatcttc tggcgtaatt ccacaattat gactatgtct 20160
tacaatagtg tacttgattt aagcaagttc aattgtaagc ataaggctac agttgttatt 20220
aatttaaagg attcatccat tagtgatgtt gtgttaggtt tgttgaagaa tggtaagttg 20280
ctagtgcgta ataatgacgc catttgtggt ttttctaatc atttggtcaa cgtaaacaaa 20340
tga 20343
Claims (8)
1. a kind of amplimer group of Porcine epidemic diarrhea virus ORF1 gene complete sequences, which is characterized in that by 8 primer pair groups
At each primer pair upstream and downstream primer is successively as shown in NO.1~16 SEQ ID.
2. a kind of amplification method of Porcine epidemic diarrhea virus ORF1 gene complete sequences, which is characterized in that include the following steps, carry
Porcine epidemic diarrhea virus sample total serum IgE is taken, cDNA is inverted to, 8 primer pairs described in claim 1 is utilized respectively and carries out PCR
Amplified reaction carries out cloning and sequencing, finally splices sequence respectively, obtains ORF1 gene complete sequences.
3. according to the method described in claim 2, it is characterized in that, 8 primer pairs be respectively primer pair ORF1-1, ORF1-2,
ORF1-3,ORF1-4,ORF1-5,ORF1-6,ORF1-7,ORF1-8；The annealing temperature of 8 primer pairs is respectively：Primer pair
ORF1-1, primer pair ORF1-6, primer pair ORF1-7, primer pair ORF1-8, annealing temperature are 62.5 DEG C；Primer pair ORF1-
2, primer pair ORF1-3, primer pair ORF1-4, primer pair ORF1-5 annealing temperature be 65 DEG C.
4. according to the method described in claim 2, it is characterized in that, the program of the pcr amplification reaction is 94 DEG C of pre-degenerations
2min；94 DEG C of denaturation 10s；Anneal 30s under annealing temperature；68 DEG C of extension 2min；Cycle 35 times, 72 DEG C extend 10min eventually；It is described
Annealing temperature corresponds to the corresponding annealing temperature of each primer pair.
5. according to the method described in claim, which is characterized in that the system of the pcr amplification reaction is that 10 μM of upstream is drawn
1.0 μ L of object, 10 μM of 1.0 μ L of downstream primer, 2 I-5 × 12.5 μ L of High Fidelity Master Mix, ddH2O
8.5 µL。
6. application of the primer sets described in claim 1 in Porcine epidemic diarrhea virus detects.
7. any one of claim 2 to 5 the method is in capture Porcine epidemic diarrhea virus variant in cell culture passages mistake
Application in journey in gene order variation.
8. application of any one of claim 2 to 5 the method in detecting pathological material of disease in Porcine epidemic diarrhea virus.
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