CN108754019B - Amplification method of porcine epidemic diarrhea virus ORF1 gene complete sequence - Google Patents
Amplification method of porcine epidemic diarrhea virus ORF1 gene complete sequence Download PDFInfo
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- CN108754019B CN108754019B CN201810532731.9A CN201810532731A CN108754019B CN 108754019 B CN108754019 B CN 108754019B CN 201810532731 A CN201810532731 A CN 201810532731A CN 108754019 B CN108754019 B CN 108754019B
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Abstract
The invention discloses an amplification method of a complete sequence of porcine epidemic diarrhea virus ORF1 gene, which relates to 8 pairs of primers, wherein the upstream primer and the downstream primer of each primer pair are shown as SEQ ID NO. 1-16 in sequence. The specific amplification step comprises the steps of extracting PEDV total RNA, inverting the PEDV total RNA into cDNA, carrying out amplification reaction by using the 8 pairs of primers, connecting a PCR product to a cloning Vector pTA2 Vector, transforming TOP10 competent cells, selecting positive clones, sequencing and identifying, and finally splicing a sequence to obtain an ORF1 genome complete sequence. The ORF1 gene of the porcine epidemic diarrhea virus variant strain in the pathological material can be obtained by amplification through the method, and the change of the gene sequence of the porcine epidemic diarrhea virus variant strain in the cell culture and passage process can be captured. The method can also be used as a method for detecting PEDV in a pathological material, and lays a foundation for the related research of ORF1 gene of PEDV.
Description
Technical Field
The invention relates to the technical field of biology, and in particular relates to an amplification method of a complete sequence of an ORF1 gene of porcine epidemic diarrhea virus.
Background
Porcine Epidemic Diarrheal Virus (PEDV) is a pathogen of Porcine Epidemic Diarrhea (PED), is a enveloped single-stranded positive-strand RNA virus, and is a member of the genus alpha coronavirus of the family Coronaviridae. The disease mainly occurs in cold tide seasons, and infected piglets die due to watery diarrhea, vomiting, dehydration and electrolyte disturbance. The severity of the disease and the mortality rate are inversely related to the day-old of the pig. Especially for newborn piglets within 7 days of age, the mortality rate can reach 100% in the absence of effective maternal antibodies. Based on the appearance and pandemic of the popular wild strains in recent years, the traditional commercial vaccines have insufficient resistance to the current popular wild strains, and the high death rate makes the pig industry economically costly.
In 1971, PED was first reported in the uk. Epidemic outbreaks of the disease are reported in succession in many countries in europe. Belgian scientists isolated coronavirus CV777 in 1976 from diarrheal pigs that developed PED. In 2005, a wild virus was isolated by scholars in China and named LJB/03. In 2006, 6 wild toxins were isolated by scholars from litter samples in the gulf of piglets. Early 2006, an outbreak of PEDV epidemic began in the immunized herd. In 2010, epidemic situations appear in China in large provinces in succession. In 10 months 2010, several provinces in the south of China developed massive PEDV infections and rapidly spread to other areas, especially the northwest. In 2011-2012, large-scale epidemic of PEDV has been outbreaked in various provinces of China.
Disclosure of Invention
The invention aims to provide an amplification method of a complete sequence of an ORF1 gene of porcine epidemic diarrhea virus, which can amplify the full length of the ORF1 gene of the porcine epidemic diarrhea virus, particularly the complete sequence of the ORF1 gene of seed viruses of different generations cultured on cells. The method can capture the sequence change of the ORF1 gene of the porcine epidemic diarrhea virus during cell culture passage, so that the correlation of the porcine epidemic diarrhea virus with virulence or immune potency can be known through genetic variation analysis of the gene.
In order to achieve the purpose, the invention is realized by the following scheme:
an amplification primer group of a complete sequence of porcine epidemic diarrhea virus ORF1 gene consists of 8 primer pairs, wherein the upstream primer and the downstream primer of each primer pair are shown as SEQ ID NO. 1-16 in sequence.
An amplification method of a complete sequence of porcine epidemic diarrhea virus ORF1 gene comprises the following steps of extracting total RNA of a porcine epidemic diarrhea virus sample, inverting the total RNA into cDNA, respectively carrying out PCR amplification reaction by using the 8 primer pairs, respectively carrying out clone sequencing, and finally splicing the sequence to obtain the complete sequence of the ORF1 gene. The total length of the target fragment ORF1 amplified by the method is 20345bp, the target fragment ORF1 consists of two large reading frames which comprise 46 nucleotide overlapping sequences and are 12,354 nucleotides and 8,037 nucleotides respectively, and the number of the occupied genome nucleotides is about 2/3.
Preferably, the procedure of the PCR amplification reaction is pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 10 s; annealing for 30s at the annealing temperature; extending for 2min at 68 ℃; circulating for 35 times, and performing final extension at 72 ℃ for 10 min; the annealing temperature corresponds to the annealing temperature corresponding to each primer pair.
Preferably, 8 primer pairs are respectively primer pairs ORF1-1, ORF1-2, ORF1-3, ORF1-4, ORF1-5, ORF1-6, ORF1-7 and ORF1-8, and the upstream and downstream primers of each primer pair are sequentially shown as SEQ ID No. 1-SEQ ID No. 16.
Preferably, the annealing temperatures of the 8 primer pairs are respectively:
the primer pair ORF1-1, the primer pair ORF1-6, the primer pair ORF1-7, the primer pair ORF1-8 and the annealing temperature are all 62.5 ℃.
Annealing of primer pair ORF1-2, primer pair ORF1-3, primer pair ORF1-4, primer pair ORF1-5
The temperatures were all 65 ℃.
Preferably, in the above method, the clone sequencing method specifically comprises recovering and purifying the amplification product, adding base A to the 3' end of the amplification product, then connecting to the cloning Vector pTA2 Vector, transforming TOP10 competent cells, screening and identifying recombinant bacteria, and then sequencing.
The specific method of the splicing sequence is as follows: the sequencing result utilizes Editseq software in Lasergene 7.10 bioinformatics software to remove the sequence at the outer end including the primer pair, and the verification or comparison search of the sequencing result is carried out by using a fast comparison tool BLAST in the GenBank in NCBI while the splicing and homology analysis of the full-length sequence is carried out, so as to complete the comparison analysis of the sequence and the existing sequence in the database.
The application of the primer group or/and the amplification method in the full-sequence amplification of the porcine epidemic diarrhea virus ORF1 gene is also within the protection scope of the invention.
The invention also provides a method for amplifying a specific region on the whole sequence of the porcine epidemic diarrhea virus ORF1 gene, which is to carry out amplification reaction by optionally combining the 8 primer pairs according to needs.
The application of the amplification method in the research of the complete sequence specific region of the porcine epidemic diarrhea virus ORF1 gene is also within the protection scope of the invention.
The amplification method can obtain the ORF1 gene of the porcine epidemic diarrhea virus variant in the pathological material. Thereby capturing the change of the gene sequence of the porcine epidemic diarrhea virus variant strain in the process of cell culture and passage. The method can also be used as a method for detecting PEDV in a pathological material, and lays a foundation for the related research of ORF1 gene of PEDV.
Compared with the prior art, the invention has the following beneficial effects:
the invention specifically designs a group of ORF3 gene complete sequences capable of cloning porcine epidemic diarrhea virus strains after 65 th generation passage. The primer group provided by the invention can amplify and obtain the ORF1 gene of the porcine epidemic diarrhea virus variant strain in the pathological material, can capture the change of the gene sequence of the porcine epidemic diarrhea virus variant strain in the cell culture and passage process, can also be used as a method for detecting PEDV in the pathological material, and lays a foundation for the related research of the ORF1 gene of the PEDV.
Drawings
FIG. 1 is an agarose gel electrophoresis chart showing the amplification result of the pair of primers designed by the present invention; m is a Marker of DL 5000; lanes 1-8 are: ORF1-1, ORF1-2, ORF1-3, ORF1-4, ORF1-5, ORF1-6, ORF1-7 and ORF 1-8.
FIG. 2 shows the position of ORF1 gene in the PEDV genome.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1
A porcine epidemic diarrhea virus ORF1 gene complete sequence amplification method comprises the following specific steps:
1. designing and synthesizing a primer: according to the nucleotide sequence of the porcine epidemic diarrhea virus variant gene disclosed in GenBank, primers are designed, and a large number of screening procedures are carried out to obtain the primer sequence of the following sequence, wherein the primer sequence and the amplification length of each primer group are shown in Table 1.
Table 1 shows the sequences of 8 primer pairs and the amplification lengths of the genes
2. Extraction of viral total RNA: adding 250 muL of virus culture solution for passaging the epidemic strains separated from the laboratory of the inventor to 65 generations on cells into 750 muL of Trizol, taking 250 muL of PBS into a sterilized centrifuge tube, adding 750 muL of TRIzol, violently oscillating, and acting for 5 minutes at room temperature. 200 muL of chloroform was added thereto, followed by vigorous shaking and standing at room temperature for 5 minutes. Centrifuge at 12,000 r/min for 15 minutes at 4 ℃. And transferring the 500 mu L of upper aqueous phase to another new sterilized microcentrifuge tube, adding 500 mu L of isopropanol, gently mixing uniformly, and placing for 15 minutes at-20 ℃. Centrifuge at 12,000 r/min for 15 minutes and decant the supernatant. 1,000 μ L of ice-chilled 75% ethanol was added and shaken up slowly by inversion. Centrifuge at 12,000 r/min for 15 minutes at 4 ℃. The supernatant was gently discarded and left for 3-5 minutes to air-dry the precipitate. Adding 20 muL RNase Free double-distilled water to dissolve RNA, and storing at-20 ℃ for later use.
3. Synthesis of first Strand of viral cDNA
The obtained cDNA was stored at-20 ℃ according to the instructions of the improved reverse transcriptase kit ReverTra Ace (TRT-101) from Toyobo (Shanghai) Biotech Co., Ltd.
4. Amplification by PCR
Amplification was performed using 8 primer pairs using the following reaction system and reaction program:
the system of the amplification reaction is 1.0 mu L of 10 mu M upstream primer and 1.0 mu L, I-52 Xhigh Fidelity Master Mix 12.5 mu L, ddH of 10 mu M downstream primer2O 8.5 µL。
The procedure of the amplification reaction is pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 10 s; annealing for 30s at the annealing temperature; extending for 2min at 68 ℃; circulating for 35 times, and performing final extension at 72 ℃ for 10 min; the annealing temperature corresponds to the annealing temperature corresponding to each primer pair.
5. Gel electrophoresis of amplification products
(1) Agarose gel preparation 2.0 g of agarose powder was weighed into a conical flask, 100 mL of electrophoresis solution was added to prepare 2% agarose gel, and the agarose gel was heated in a microwave oven to dissolve the agarose powder sufficiently. And then taking out and placing in a normal-temperature water tank to cool to about 50 ℃, adding 7 mu L Gold View nucleic acid dye, and fully and uniformly mixing. The gel was poured and allowed to set after about 20 minutes at room temperature.
(2) Nucleic acid electrophoresis, adding the PCR product to the gel well, and adding 5 μ L of DNA Marker DL5,000 as a control. And performing electrophoresis at the voltage of 120V for 30-40 minutes. And observing the result on a gel image processing system and taking a picture for storage.
6. And (3) recovering and purifying amplification products: all amplification products were subjected to agarose gel electrophoresis, and the target fragments were excised under brief irradiation of an ultraviolet lamp and recovered and purified with reference to a fast recovery kit for DNA from magenta.
7. Adding a base A at the 3' end of a gel recovery product: the fragment amplified by the high fidelity enzyme is blunt-ended, and the common enzyme has the characteristic of adding one base A at the 3' end of the product, so that the fragment can be connected with a pTA2 Vector at the cohesive end, and the efficiency of connecting the fragment with the pTA2 Vector can be greatly improved. The specific reaction system is shown in Table 2.
Table 2 shows the reaction system of adding base A to the 3' end of the recovered product:
the reaction program was 72 ℃ for 30 minutes.
8. And (3) connecting the target fragment with a vector: the product of base A added to the 3' end was subjected to concentration measurement, and ligated to the vector in an amount of 125ng, and the ligation system is shown in Table 3.
Table 3 is the attachment system:
the reaction mixture was reacted at room temperature (15 to 25 ℃) for 30 minutes.
9. Ligation products transformed competent cells: the component Cell (100. mu.l) was thawed in an ice bath. Add 10. mu.l of ligation mix gently and place in ice bath for 30 min. After 30 seconds heat shock at 42 ℃, the mixture was cooled in an ice bath for 2 minutes. Adding 900 muL of SOC culture medium, and performing shake culture at 37 ℃ for 1 hour. An appropriate amount was smeared on an LB plate and cultured overnight at 37 ℃.
10. Determination of recombinants: the method comprises the steps of picking suspected positive single colonies with uniform size and round shape from an LB agar plate, picking each colony to be detected into a 1.5 mL sterile centrifuge tube of an LB liquid culture medium containing 30 mu L ampicillin through aseptic operation, blowing and beating the colonies uniformly by a pipette to form bacterial suspension, and carrying out colony PCR identification by taking the suspension as a template, wherein the sequence of a primer pair is shown in Table 1. The amplification and identification of each gene segment are carried out by using corresponding primers respectively, and the system is shown in Table 4.
Table 4 PCR system for colony identification:
| 2xTaq PCR Master Mix | 10.0μL |
| P1(10μM) | 0.5μL |
| P2(10μM) | 0.5μL |
| bacterial colony template | 1.0μL |
| ddH2O | 8.0μL |
| Total | 20.0μL |
PCR amplification procedure:
and (3) carrying out agarose gel electrophoresis on the amplified product, selecting corresponding bacterial liquid capable of amplifying strong and specific target bands, shaking the bacterial liquid into a liquid culture solution containing the ampicillin, and sequencing the bacterial liquid by Jinzhi biotechnology limited.
11. Processing of Gene sequencing results
The sequencing result utilizes Editseq software in Lasergene 7.10 bioinformatics software to remove the sequence at the outer end including the primer pair, and the verification or comparison search of the sequencing result is carried out by using a fast comparison tool BLAST in the GenBank in NCBI while the splicing and homology analysis of the full-length sequence is carried out, so as to complete the comparison analysis of the sequence and the existing sequence in the database. The complete sequence of the gene ORF3 of the virus of the 65 th generation of the epidemic strain cell passage is shown as SEQ ID NO: shown at 17.
Sequence listing
<110> southern China university of agriculture
<120> amplification method of porcine epidemic diarrhea virus ORF1 gene complete sequence
<160> 17
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gagaagttta aagtcttcct taactgtgt 29
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caacaacagg tttgttgtct gtgacaacg 29
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cacgtagtgt taaggctgtt aaagtagaa 29
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atgaaggtta tacccttaac tttagtcgt 29
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ggaagaaact aggaagtaca ttatccgta 29
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ctaattctga ccaaaagaaa gtcagaaaa 29
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gcgcctgcag aaatgtcttg ctggtt 26
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cgaacacagt tcaccttgag gaatttacc 29
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ctacaacaag gatgttgctt gtctaggta 29
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cagatgctta acccaatcca aaagcacat 29
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gaaaaccctg aatataagaa ggtgtttta 29
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tgtcaactcc aaaccaccag cccacaaca 29
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ctacctggcc aacctatcag acatactaa 29
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tcatttgttt acgttgacca aatgattaga aaa 33
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<213> porcine epidemic diarrhea virus (porcine epidemic diarrhea virus)
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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 (4)
1. A method for amplifying the complete sequence of the ORF1 gene of porcine epidemic diarrhea virus, said method being a method for non-diagnostic purposes, comprising the steps of: extracting total RNA of a porcine epidemic diarrhea virus sample, inverting the total RNA into cDNA, respectively carrying out PCR amplification reaction by using 8 primer pairs, respectively carrying out clone sequencing, and finally splicing the sequence to obtain an ORF1 gene complete sequence;
the sequences of the upstream primers and the downstream primers of the 8 primer pairs are sequentially shown as SEQ ID No. 1-16;
the 8 primer pairs are respectively primer pairs of ORF1-1, ORF1-2, ORF1-3, ORF1-4, ORF1-5, ORF1-6, ORF1-7 and ORF 1-8; the annealing temperatures of the 8 primer pairs were: the annealing temperatures of the primer pair ORF1-1, the primer pair ORF1-6, the primer pair ORF1-7 and the primer pair ORF1-8 are all 62.5 ℃; the annealing temperatures of the primer pair ORF1-2, the primer pair ORF1-3, the primer pair ORF1-4 and the primer pair ORF1-5 are all 65 ℃;
the procedure of the PCR amplification reaction is as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 10 s; annealing for 30s at the annealing temperature; extending for 2min at 68 ℃; circulating for 35 times, and performing final extension at 72 ℃ for 10 min; the annealing temperature is the annealing temperature corresponding to each primer pair.
2. The method of claim 1, wherein the PCR amplification reaction is performed in a system comprising: 10 mu M of upstream primer 1.0 mu L, 10 mu M of downstream primer 1.0 mu L, 2 Xhigh Fidelity Master Mix 12.5 mu L, ddH2O8.5 µL。
3. Use of the method of any one of claims 1 to 2 to capture genetic sequence changes in a variant porcine epidemic diarrhea virus during cell culture passaging.
4. Use of the method of any one of claims 1 to 2 for detecting porcine epidemic diarrhea virus in a patient.
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