CN109593892A - A kind of universal primer group, kit and method expanding H5N6 subtype avian influenza virus full-length genome - Google Patents
A kind of universal primer group, kit and method expanding H5N6 subtype avian influenza virus full-length genome Download PDFInfo
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Abstract
The invention discloses a kind of universal primer group, kit and methods for expanding H5N6 subtype avian influenza virus full-length genome, the primer sets of design carry out One step RT-PCR amplification, 12 pairs of all systems of primer are consistent, annealing temperature is consistent, amplification condition is consistent, the method eliminates the operation of the most suitable annealing temperature of primer one, keep operating method easier, it is time saving, laborsaving.Same response procedures can expand several genes segment simultaneously, can be quickly obtained the full-length genome sequence of H5N6 subtype avian influenza virus, the amplification H5N6 subtype avian influenza virus full-length genome time is shorter, 1.5 days available full-length genomes.With the advantages that simplicity, time-consuming is few, save the cost.
Description
Technical field
The present invention relates to viral genome amplifications, and in particular to a kind of amplification H5N6 subtype avian influenza virus full-length genome
Universal primer, kit and method.
Background technique
Bird flu is the abbreviation of Bird Flu, is a kind of birds (poultry and wild fowl) as caused by influenza A
Infectious disease.Avian influenza virus (Avian Influenza Virus, AIV) infection after can show as slight respiratory symptom,
Symptom of digestive tract.According to avian influenza virus is pathogenic and the difference of virulence, bird flu can be divided into highly pathogenic bird flu, low
Pathogenic avian influenza and no pathogenicity bird flu.Avian influenza virus is tunicary RNA virus, and form is irregular, mode of appearance
For the spherical of 80~120nm of diameter or up to the filiform of thousands of nanometers, genome is made of 8 sub-thread strand RNAs, is encoded altogether
10 kinds of protein, including hemagglutinin (Hemagglutinin, HA), neuraminidase (Neuraminidase, NA), nucleoprotein
It is (Nucleoprotein, NP), stromatin (Stroma protein, SP), polymerase protein (PB1, PB2, PA), non-structural
Albumen (Nonstructural proteins, NS) and other protein (HE, M2, M3, NB).
In recent years, highly pathogenic avian influenza virus constantly makes a variation in field, 2013~2014 years, seminar Henan,
When the area such as Hebei, Guangdong, Guangxi, Jiangsu, Fujian, Shandong, Yunnan carries out avian influenza virus epidemiological survey, it is isolated to
HA gene order is located at the new evolutionary branching of 2.3.4, Ji Lei 2.3.4 branch, which has become the main stream in the area such as south China
Row strain, 2014~2015 year H5 subtype avian influenza epidemiological surveys the result shows that, class " clade 2.3.4 " genotype
The increase of strain quantity is most fast, pollution face is most wide, and a variety of hypotype strains such as H5N1, H5N2, H5N6 and H5N8 coexist, and make to aviculture
At biggish loss, the strain of " clade 2.3.4 " genotype brings about great losses there is no the gesture of decrease to aviculture.
H5 subtype avian influenza epidemiological survey in 2016~2018 years the result shows that, clade 2.3.4 is branched off into Major Epidemic branch,
Up to now, existing H5N6 subtype avian influenza virus belongs to the branch, therefore research can be quickly obtained H5N6 subtype avian influenza
The whole genome sequence of virus is most important for the investigation and analysis of bird flu epidemiology and safety control of bird flu.
The genome sequencing of existing H5N6 subtype avian influenza virus has the disadvantage in that traditional laboratory carries out gene
Cloning and sequencing is that DNA fragmentation is connected in carrier, then converts to competent bacteria, then filters out positive colony bacterium progress
Expand numerous, extraction bacteria plasmid DNA after expansion is numerous, then is sent to professional sequencing company and is sequenced.The method is time-consuming, at high cost, efficiency
It is low, be not suitable for large batch of sequencing, this method is difficult to find hybrid virus infection, the full-length genome sequence for being easy to make mistake
Column.
Summary of the invention
One of the objects of the present invention is to provide a kind of universal primers for expanding H5N6 subtype avian influenza virus full-length genome
Group.
Another object of the present invention is to provide a kind of kits for expanding H5N6 subtype avian influenza virus full-length genome.
A further object of the present invention is to provide a kind of methods for expanding H5N6 subtype avian influenza virus full-length genome
Another object of the present invention is to provide a kind of sequencing approach of H5N6 subtype avian influenza virus full-length genome.
The technical solution used in the present invention is:
A kind of universal primer group expanding H5N6 subtype avian influenza virus full-length genome, nucleotide sequence are as follows:
Primer pair 1:
HA1-F:AGCARAAGCAGGG (SEQ ID NO:1)
HA1-R:TCTGCTCAATAGGTGTT (SEQ ID NO:2)
Primer pair 2:
HA2-F:CCAAAATRAACGGGCA (SEQ ID NO:3)
HA2-R:AGTAGAAACAAGGGTGR (SEQ ID NO:4)
Primer pair 3:
NA-F:AGGGTGAAAATGAAT (SEQ ID NO:5)
NA-R:CTCCTGTTGCTGAAATGCA (SEQ ID NO:6)
Primer pair 4:
M-F:TCTTCTAACCGAG (SEQ ID NO:7)
M-R:TTCTATGTTGACAAA (SEQ ID NO:8)
Primer pair 5:
NS-F:ACACTGTGTCAAG (SEQ ID NO:9)
NS-R:AAGCTGAAACGAGAAGG (SEQ ID NO:10)
Primer pair 6:
NP-F:CGATCTTATGAACAGA (SEQ ID NO:11)
NP-R:CTGCATTGTCTCCGAAG (SEQ ID NO:12)
Primer pair 7:
PA1-F:AGCRAAAGCAGGTACTGATY (SEQ ID NO:13)
PA1-R:CATNCCCATCATCAT (SEQ ID NO:14)
Primer pair 8:
PA2-F:ACATTTTTCGGCTGGAAA (SEQ ID NO:15)
PA2-R:GAAACAAGGCATTTT (SEQ ID NO:16)
Primer pair 9:
PB11-F:GAGACTGGAGCACCYCAACT (SEQ ID NO:17)
PB11-R:AGCRAAAGCAGGCAAACY (SEQ ID NO:18)
Primer pair 10:
PB12-F:CRAAAGCAGGTCAAWTATAT (SEQ ID NO:19)
PB12-R:ACAAGGTCGTTTTWAAACW (SEQ ID NO:20)
Primer pair 11:
PB21-F:ARTTCACAATGGTTGG (SEQ ID NO:21)
PB21-R:ACAAGGCATTTTTTCA (SEQ ID NO:22)
Primer pair 12:
PB22-F:TAATAAAGCTGCTACC (SEQ ID NO:23)
PB22-R:CAGCAGGTYCCTT (SEQ ID NO:24).
A kind of kit expanding H5N6 subtype avian influenza virus full-length genome, the kit contain primer described above
Group.
Preferably, mentioned reagent box also contains dNTP, Taq enzyme, reverse transcriptase.
A method of amplification H5N6 subtype avian influenza virus full-length genome, comprising the following steps:
1) sample rna is extracted, is cDNA by RNA reverse transcription；
2) using cDNA as template, RT-PCR amplification is carried out respectively with 12 pairs of primer pairs described above, obtains H5N6 hypotype fowl
The amplified production of influenza virus full-length genome.
Preferably, RT-PCR amplification system is in step 2)
Preferably, RT-PCR amplification system program in step 2) are as follows: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 50S, 58 DEG C move back
Fiery 30S, 72 DEG C of extension 3min, 35 circulations, 72 DEG C of extension 5min are carried out.
A kind of sequencing approach of H5N6 subtype avian influenza virus full-length genome is obtained according to method described in any of the above embodiments
The amplified production for obtaining H5N6 subtype avian influenza virus full-length genome, amplified production is sequenced with full-automatic sequence instrument
And splicing is to get the nucleotide sequence of H5N6 subtype avian influenza virus full-length genome.
The beneficial effects of the present invention are:
(1) primer sets designed carry out One step RT-PCR amplification, and 12 pairs of all systems of primer are consistent, annealing temperature one
It causes, amplification condition is consistent, and the method eliminates the operation of the most suitable annealing temperature of primer one, keeps operating method simpler
Just, time saving, laborsaving.Same response procedures can expand several genes segment simultaneously, can be quickly obtained H5N6 subtype avian influenza disease
The full-length genome sequence of poison, the amplification H5N6 subtype avian influenza virus full-length genome time is shorter, 1.5 days available full genomes
Group.With the advantages that simplicity, time-consuming is few, save the cost.
(2) it is sequenced using segmentation primer, keeps the Sequence of overlapping more conservative, it is ensured that the accuracy of column is sequenced.
(3) the higher sequence of accuracy rate can be obtained using PCR product sequencing, reduces to clone there are product recycling and TA and surveys
The work of sequence.
(4) column signal peak be sequenced does not occur covering peak miscellaneous peak, and clean background, sequencing result is accurate, can be used for distinguishing poison
Whether strain has the case where mixed infection.
(5) present invention can be applied to all H5N6 subtype avian influenza virus whole genome amplifications, both include the Asia H5N6
The extremely conservative noncoding region of the genome of type avian influenza virus and its both ends, applicability is wide, and versatility is good, and is suitble to large quantities of
It measures sequence and verifies whether mixed infection.
Detailed description of the invention
The sequencing peak of Fig. 1 H5N6 subtype avian influenza virus GDJM plants of HA genes of prevalence strain；
Fig. 2 is the electrophoresis detection figure of H5N6 sequencing primer gene RT-PCR amplified production of the present invention；M is DL2000；1:
HA1；2:HA2；3:NA；4:M；5:NS；6:NP；7:PA1；8:PA2；9:PB11；10:PB12；11:PB21；12:PB22.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The design and synthesis of embodiment 1:H5N6 subtype avian influenza virus genome sequencing primer
Download the CHINESE REGION 2010-2016 and China national influenza center virus sequence data in GenBank database
HA, NA, PA, PB1, PB2, NP, M and NS gene of H5N6 subtype avian influenza virus in library, software compare analysis different virus and
Different genes sequence selects opposite conserved regions design full-length genome specificity amplification primer sequence.Allow in design of primers same
Variant sites allow 4 and 4 or less degeneracy bases.Designed primer is largely screened and verified, is finally imitated
The optimal one group of primer of fruit, as shown in table 1, this group of primer include 12 pairs of primers, which carries out One step RT-PCR expansion
Increase, 12 pairs of all reaction systems of primer are consistent, annealing temperature is consistent, and amplification condition is consistent, and same response procedures can be simultaneously
Several genes segment is expanded, the operation of the most suitable annealing temperature of primer one is eliminated, keeps operating method easier, is saved
When, it is laborsaving, the full-length genome sequence of H5N6 subtype avian influenza virus can be quickly obtained, there is simplicity, time-consuming is few, save the cost
The advantages that.
1 H5N6 subtype avian influenza virus genome sequencing primer sets of table
| Primer | Primer sequence |
| HA1-F | AGCARAAGCAGGG(SEQ ID NO:1) |
| HA1-R | TCTGCTCAATAGGTGTT(SEQ ID NO:2) |
| HA2-F | CCAAAATRAACGGGCA(SEQ ID NO:3) |
| HA2-R | AGTAGAAACAAGGGTGR(SEQ ID NO:4) |
| NA-F | AGGGTGAAAATGAAT(SEQ ID NO:5) |
| NA-R | CTCCTGTTGCTGAAATGCA(SEQ ID NO:6) |
| M-F | TCTTCTAACCGAG(SEQ ID NO:7) |
| M-R | TTCTATGTTGACAAA(SEQ ID NO:8) |
| NS-F | ACACTGTGTCAAG(SEQ ID NO:9) |
| NS-R | AAGCTGAAACGAGAAGG(SEQ ID NO:10) |
| NP-F | CGATCTTATGAACAGA(SEQ ID NO:11) |
| NP-R | CTGCATTGTCTCCGAAG(SEQ ID NO:12) |
| PA1-F | AGCRAAAGCAGGTACTGATY(SEQ ID NO:13) |
| PA1-R | CATNCCCATCATCAT(SEQ ID NO:14) |
| PA2-F | ACATTTTTCGGCTGGAAA(SEQ ID NO:15) |
| PA2-R | GAAACAAGGCATTTT(SEQ ID NO:16) |
| PB11-F | GAGACTGGAGCACCYCAACT(SEQ ID NO:17) |
| PB11-R | AGCRAAAGCAGGCAAACY(SEQ ID NO:18) |
| PB12-F | CRAAAGCAGGTCAAWTATAT(SEQ ID NO:19) |
| PB12-R | ACAAGGTCGTTTTWAAACW(SEQ ID NO:20) |
| PB21-F | ARTTCACAATGGTTGG(SEQ ID NO:21) |
| PB21-R | ACAAGGCATTTTTTCA(SEQ ID NO:22) |
| PB22-F | TAATAAAGCTGCTACC(SEQ ID NO:23) |
| PB22-R | CAGCAGGTYCCTT(SEQ ID NO:24) |
Since the length of part influenza virus gene segment upstream and downstream conserved region is different, lead to individual upstream and downstream primer length
It differs greatly, primer pair Tm value difference is different big, and conventional One step RT-PCR is difficult to effectively expand；The present invention is flowed for H5N6 hypotype fowl
8 genetic fragments of Influenza Virus, design in same response procedures while expanding, and substantially increase amplification efficiency, one-step method RT-
PCR directly send PCR product to be sequenced.
The response procedures of 8 genetic fragments are as follows: be first cDNA, 50~55 DEG C of reverse transcriptions by the sample rna reverse transcription of extraction
20~40min；RT-PCR, RT-PCR response procedures are carried out by template of cDNA again are as follows: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
50S, 58 DEG C of annealing 30S, 72 DEG C of extension 3min carry out 35 circulations, 72 DEG C of 4 DEG C of postposition of extension 5min preservations.
The full genome of GDJM plants of prevalence strain of embodiment 2:H5N6 subtype avian influenza virus expands and sequencing result analysis
Method:
1) all dead to be inoculated with 37 DEG C of strain of prevalence strain GDJM of H5N6 subtype avian influenza virus cultures 48 hours, harvest is dead
The RNA of the avian influenza virus harvested in SPF chicken embryo after dying, and be cDNA as template using the RNA reverse transcription；
2) it is template that cDNA is walked more than, utilizes PCR amplification primer sets HA1-F, HA1-R, HA2- of avian flu virus gene
F、 HA2-R、NA-F、NA-R、M-F、M-R、NS-F、NS-R、NP-F、NP-R、PA1-F、PA1-R、PA2-F、 PA2-R、PB11-
F, totally 12 pairs of primers carry out respectively by PB11-R, PB12-F, PB12-R, PB21-F, PB21-R, PB22-F, PB22-R (being shown in Table 1)
RT-PCR amplification；PCR product is sequenced and is spliced with full-automatic sequence instrument.
The system of above-mentioned RT-PCR amplified reaction is as shown in table 2.Above-mentioned RT-PCR amplification program are as follows: 95 DEG C of initial denaturations
5min, 95 DEG C of denaturation 50S, 58 DEG C of annealing 30S, 72 DEG C of extension 3min, 35 circulations, 72 DEG C of 4 DEG C of postposition of extension 5min guarantors are carried out
It deposits.
The system of 2 RT-PCR amplified reaction of table
| Serial number | Constituent | Dosage |
| 1 | Buffer | 5μL |
| 2 | 2.5mM dNTP | 4μL |
| 3 | 10pmol/ μ L upstream primer | 2μL |
| 4 | 10pmol/ μ L downstream primer | 2μL |
| 5 | cDNA | 1.5μL |
| 6 | Ex Taq | 0.25μL |
| 7 | ddH2O | 35μL |
As a result:
The sequencing peak figure of HA gene is not as shown in Figure 1, signal peak occurs covering peak miscellaneous peak, clean background, sequencing result standard
Really.Sequencing result such as NO:25~32 SEQ ID for the full genome amplification that GDJM plants of prevalence strain of H5N6 subtype avian influenza virus
Shown, i.e., HA gene order is SEQ ID NO:25, NA gene order is SEQ ID NO:26, M gene order is SEQ ID
NO:27, NS gene order are SEQ ID NO:28, NP gene order is SEQ ID NO:29, PA gene order is SEQ ID
NO:30, PB1 gene order are SEQ ID NO:31, PB2 gene order is SEQ ID NO:32.
Embodiment 3: detection H5N6 subtype avian influenza virus positive pathological material of disease and sequencing
1) 200 μ l tissue fluid (animal's liver, spleen, pancreas, brain tissue etc.), 1 μ l the extraction of viral RNA: are taken
Carrier RNA, 20ul Proteinase K, 200ul Buffer VGB, mixes well, in 56 DEG C of constant temperature warm bath 10min；
This presses Takara RNA extraction agent box specification extraction viral RNA to tool, and is cDNA by the RNA reverse transcription.
2) using above-mentioned cDNA as template, PCR amplification primer sets HA1-F, HA1-R, HA2- of avian flu virus gene are utilized
F、 HA2-R、NA-F、NA-R、M-F、M-R、NS-F、NS-R、NP-F、NP-R、PA1-F、PA1-R、PA2-F、 PA2-R、PB11-
F, totally 12 pairs of primers carry out respectively by PB11-R, PB12-F, PB12-R, PB21-F, PB21-R, PB22-F, PB22-R (being shown in Table 1)
RT-PCR amplification；PCR product is sequenced and is spliced with full-automatic sequence instrument.
The system of above-mentioned RT-PCR amplified reaction is as shown in table 2, and the reaction tube for having added reaction system is put in PCR instrument and is carried out
RT-PCR, response procedures are as follows: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 50S, 58 DEG C of annealing 30S, 72 DEG C of extension 3min, carrying out
35 circulations, 72 DEG C of 4 DEG C of postposition of extension 5min preservations.
3) RT-PCR product detection: respectively take 6 × loading Buffer of 5uL product and 1uL in 1%Agrorose gel
It carries out electrophoresis and observes result.
As a result: for testing result as shown in Fig. 2, 12 reaction products obtain specific band, amplification obtains H5N6 hypotype
Avian influenza virus full-length genome, and success is sequenced, illustrate that 12 in the embodiment of the present invention pairs of primers can be expanded efficiently and accurately
H5N6 subtype avian influenza virus full-length genome out.The present invention also has chosen 2016~2018 years popular H5N6 subtype avian influenzas
It 66 plants of virus HN XY, SDZC, GDHZ, GDQY, JSXZ, FJZZ, SDLW, JSPX, SCCD etc., is carried out using 12 pairs of primers of the present invention
Whole genome amplification, 792 reactions all obtain such as Fig. 2 similar positive amplification product, and are sequenced and succeed, and illustrate the present invention
12 pairs of primers have good versatility.
In conclusion the primer sets that (1) present invention designs carry out One step RT-PCR amplification, all systems one of 12 pairs of primers
Cause, annealing temperature are consistent, and amplification condition is consistent, and the method eliminates the operation of the most suitable annealing temperature of primer one, makes to grasp
It is easier to make method, it is time saving, laborsaving.Same response procedures can expand several genes segment simultaneously, can be quickly obtained H5N6
The full-length genome sequence of subtype avian influenza virus, the amplification H5N6 subtype avian influenza virus full-length genome time is shorter, 1.5 days can
Obtain full-length genome.With the advantages that simplicity, time-consuming is few, save the cost.
(2) it is sequenced using segmentation primer, keeps the Sequence of overlapping more conservative, it is ensured that the accuracy of column is sequenced.
(3) the higher sequence of accuracy rate can be obtained using PCR product sequencing, reduces to clone there are product recycling and TA and surveys
The work of sequence.
(4) column signal peak be sequenced does not occur covering peak miscellaneous peak, and clean background, sequencing result is accurate, can be used for distinguishing poison
Whether strain has the case where mixed infection.
(5) present invention can be applied to all H5N6 subtype avian influenza virus whole genome amplifications, both include the Asia H5N6
The extremely conservative noncoding region of the genome of type avian influenza virus and its both ends, applicability is wide, and versatility is good, and is suitble to large quantities of
It measures sequence and tests and prove whether mixed infection.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou City Huanan Nonda Biology Medicine Co., Ltd
<120>a kind of universal primer group, kit and method for expanding H5N6 subtype avian influenza virus full-length genome
<130>
<160> 32
<170> PatentIn version 3.5
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ggaaatttta ttgctccaga atatgcatac aaaattgtca agaaagggga ctcaacaatc 840
atgaaaagtg aaatggaata tggccattgc aacaccaaat gtcaaactcc aataggggcg 900
ataaactcta gtatgccatt ccacaatata caccctctca caatcgggga atgccccaaa 960
tacgtgaaat caaacaaatt agtacttgcg actgggctca gaaatagtcc tctaagggag 1020
aggagaagaa aaagaggact atttggagct atagcagggt ttatagaggg aggatggcaa 1080
ggaatggtag atggttggta tgggtaccac catagcaatg aacagggaag tgggtacgct 1140
gcagacaaag aatccaccca aaaggcaata gatggagtta ccaataaggt caactcgatc 1200
attgacaaga tgaacactca atttgaggcc gttggaaggg aatttaataa cttagaacgg 1260
agaatagaga atttaaacaa gaaaatggaa gacggattcc tagatgtctg gacttataat 1320
gcggaacttc tagttctcat ggaaaatgag agaaccctag atttccatga ctcaaatgtc 1380
aagaaccttt atgacaaagt ccgactacag cttagggata atgcaaagga gctgggtaat 1440
ggttgctttg agttctatca caaatgtgat gatgaatgta tggaaagtgt aagaaatggg 1500
acatatgact accctcagta ttcagaagaa gcaagattga aaagagaaga aataagcgga 1560
gtgaaattgg aatcaatagg aacttaccag atactgtcaa tttattcaac agtggcgagt 1620
tccctagcac tggcaatcat tgtggctggt ctatctttat ggatgtgctc caatgggtca 1680
ttacaatgca gaatttgcat ttga 1704
<210> 26
<211> 1380
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 26
atgaatccaa atcaaaagat agcatgcatt tcagcaacag gagtaacact atcggtggta 60
agcctgctaa taggaattgc caatttgggc ctaaatatcg ggctacacta caaagtgaat 120
aattcaacaa ctataaacat tccaaacatg aatgagacca acccaacaac aacaaacatc 180
accaatatta tagtgaataa gaacgaagaa agaacatttc tcaacttgac caagccgcta 240
tgtgaagtca actcatggca cattctatcg aaagacaatg caataagaat aggtgaggat 300
gctcatatac tgatcacaag ggaaccttac ttgtcctgtg atccacaagg atgcaggatg 360
tttgctctga gtcaaggcac aacactcaga gggcgacatg cgaatggaac catacatgat 420
aggagcccat ttcgagctct cataagttgg gaaatgggtc aggcacccag tccatataac 480
actagggtcg aatgcatagg atggtcaagc acgtcatgcc atgatggcat atcaaggatg 540
tcaatatgca tatcaggacc gaataacaat gcctcggcag tggtgtggta cagggggcga 600
ccagtaacag aaatcccatc atgggtaggg aacattctta ggactcaaga atcagagtgt 660
gtgtgccata aaggaatctg cccagtagtt atgacagatg gtccagctaa caacaaggca 720
gcaactaaga taatctactt caaagaggga aagatacaga aaattgaaga actgcaaggg 780
aacgctcaac acatcgaaga gtgttcatgc tacggagcag cagggatgat caaatgtgta 840
tgcagagaca attggaaggg ggcaaataga ccaataatca ctatagatcc cgaaatgatg 900
acccacacaa gcaaatactt gtgttcaaaa atcttaaccg acacaagccg tcctaatgac 960
cccatcaatg ggaactgcga tgcgccaata acaggaggga gcccagaccc aggggtaaaa 1020
gggtttgcat tcctagacgg ggagaattca tggcttggaa ggacaattag taaagactcc 1080
agatcaggct acgaaatgtt aaaggtccca aatgcagaaa ccgacactca atcagggcca 1140
acctcatacc agctgattgt caacaatcaa aattggtcag ggtactcagg ggcattcata 1200
gactactggg cgaacaggga atgcttcaat ccttgttttt acgtggagct aatcagaggg 1260
agacccaaag agattgatgt actgtggact tccaatagca tggttgctct ctgtggatcc 1320
agggagcgat tgggatcatg gtcctggcat gatggtgcag aaatcatcta ctttaagtag 1380
<210> 27
<211> 989
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 27
atgagtcttc taaccgaggt cgaaacgtac gttctctcta tcatcccatc aggccccctc 60
aaagccgaga tcgcgcagaa acttgaggat gtgtttgcag gaaagaacgc tgatctcgag 120
gctctcatgg agtggctaaa gacaagacca atcctgtcac ctctgactaa aggaattttg 180
ggatttgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acggtttgtc 240
cagaatgccc taaatggaaa tggagatcca aataatatgg atagggcagt taagctatat 300
aaaaagctga aaagagaaat aacatttcat ggagctaagg aggtcgcact cagttactca 360
actggtgcac ttgccagttg catgggtctc atatacaaca gaatgggaac agtgactaca 420
gaagtggctt ttggcctagt gtgtgccact tgtgagcaga ttgcggattc acagcatcgg 480
tctcacagac agatggcaac catcaccaac ccactaatca ggcatgagaa cagaatggtg 540
ctggccagca ctacagctaa ggctatggag cagatggcgg gatcaagtga gcaggcagca 600
gaagccatgg aggtcgccaa tcaggctaga cagatggtgc aggcaatgag aacaattggg 660
actcatccta attctagtac tggtctgaga gacaatcttc ttgaaaattt gcaggcctac 720
cagaaacgga tgggagtgca gatgcagcga ttcaagtgat cctcttgttg ttgccgcaag 780
tatcattggg atcttgcact tgatattgtg gattcttgat cgtcttttct tcaaatgcat 840
ttatcgtcgc cttaaatacg gtttgaaaag agggccttct acggaaggag taccggagtc 900
tatgagggaa gagtaccggc aggaacagca gaatgctgta gatgttgacg atggtcattt 960
tgtcaacata gagttggagt aaaaaacta 989
<210> 28
<211> 845
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 28
atggattcca acactgtgtc aagttttcag gtagactgct ttctttggca tgtccgcaaa 60
cgatttgcag accaagaact gggtgatgcc ccattccttg accggcttcg ccgagatcag 120
aagtccctaa gaggaagagg caacactctt ggtctggaca tcgaaacagc tactcgtgcg 180
ggaaaacaaa tagtggagcg gattcttgat gaggaacctg atgaagcact taaaatgccg 240
acttcacgtt acctaactga aatgactctc gaagaaatgt cgagggactg gttcatgctc 300
atgcccaagc agaaagtggt gggttccctt tgcatcaaaa tggaccaggc aataatggat 360
aaaagcatca tactgaaagc aaatttcagt gtgatttttg gccggttaga gaccctaata 420
ctgctcagag ctttcacaga agaaggagca atcgtgggag aaatctcacc attaccttct 480
cttccaggac atactggcga ggatgtcaaa aatgcaattg gcgtcctcat cggaggactt 540
gaatggaatg ataacacagt tcgggtctct gaagttatac agagattcgc ttggagaagc 600
agtgatgagg gtgggagact tccactccct ccaaatcaga aacggaaact ggcgagagca 660
attgagtcag aagtttgaag aaataaggtg gctgattgaa gaggtacgac atagattgaa 720
aattacagaa aacagcttcg aacagataac ttttatgcaa gccttacaac tactgcttga 780
agtggagcaa gagataagag ccttctcgtt tcagcttatt taatgataaa aaacaccttt 840
gtggt 845
<210> 29
<211> 1497
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 29
atggcgtctc aaggcaccaa acgatcttat gaacagatgg aaactggtgg agagcgccag 60
aatgctactg agatcagggc ctctgttgga agaatggttg gtggcattgg gaggttctac 120
atacagatgt gcacagaact caaactcagc gaccatgaag ggagactgat ccagaacagc 180
ataacaatag agagaatggt actttctgca tttgatgaaa gaaggaacag gtatctggaa 240
gagcacccca gtgcagggaa ggaccctaag aaaactggag gtccaattta tcggaggaga 300
gacgggaaat ggattagaga gctgattttg tacgacaaag aagagatcag gaggatttgg 360
cgccaagcaa acaacggaga ggacgcaact gctggtctta cccacctgat gatatggcac 420
tccaatctga atgatgccac atatcagaga acaagagctc tcgtgcgtac cggaatggac 480
cccaggatgt gctccctaat gcagggatca actcttccga gaagatctgg agctgctggt 540
gcagcagtga agggggtagg aacaatggtg atggagctgg ttcgaatgat aaaacgaggg 600
attaacgacc ggaatttctg gagaggcgaa aatggacgga gaacaaggat tgcatatgag 660
agaatgtgca acatcctcaa agggaaattc caaacagctg cacaaagagc aatgatggat 720
caagtgcgag agagcaggaa tcctgggaat gctgaaattg aagatcttat ttttctggca 780
cggtctgcac tcatcctgag aggatcagtg gcccataagt cctgcttgcc tgcttgtgtg 840
tacggacttg cagtggccag tgggtatgat ttcgagagag aaggatactc tctggttggg 900
atagatcctt tccgtttgct tcaaaacagc caggtcttta gtctcattag gccaaatgaa 960
aacccagcac ataagagtca attagtgtgg atggcatgcc actctgcagc atttgaggat 1020
ctcagagtct caagtttcat cagagggaca agagttgtcc caagagggca gctatccact 1080
agaggggttc aaattgcttc aaatgagaac atggaaacaa tggactccaa cacacttgag 1140
ctgagaagta gatattgggc tataagaacc aggagcggag ggaataccaa ccagcagagg 1200
gcatctgcag ggcagattag tgttcaaccc actttctcgg tgcagagaaa ccttcccttc 1260
gaaagagcga ccattatggc ggcatttgca ggaaatactg aaggcagaac gtccgacatg 1320
aggacagaaa tcataagaat gatggaaaat gccaaaccag aagatgtgtc attccagggg 1380
cggggagtct tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac 1440
atgaataatg aaggatctta tttcttcgga gacaatgcag aggagtatga caattaa 1497
<210> 30
<211> 2172
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 30
atggaggact ttgtgcgaca tgcttcatcc aatgatcgtc gagcttgcgg aaaagacaat 60
gaaagaatat ggggaaagtc cgaaaatcga gacgaacaaa tttgccgcaa tatgcacaca 120
cctagaagtc tgcttcatgt actcggactt ccactttata gatgaacgag gcgaatcaat 180
aattgcagag tctggcgatc cgaacgcatt attgaaacac cgatttgaga taattgaagg 240
gagggaccga acaatggctt ggacagtggt aaacagtatc tgcaacacca caggggtcga 300
taagcctaaa ttcctcccag atttatatga ctacaaagag aaccggttta ttgaaattgg 360
agtgacacgg agggaagtcc acatatacta cctagaaaaa gcaaataaga taaaatcaga 420
gaggacacac atccacatat tctcattcac tggagaggaa atggccacta aagctgacta 480
tactcttgat gaggagagca gagcaagaat taaaacccgg ctgttcacta taagacaaga 540
aatggccagt aggggtctat gggattcctt tcgtcaatcc gagagaggcg aagagacaat 600
tgaagaaagg tttgaaatca caggaaccat gcgcaggctt gctgaccaaa gtctcccacc 660
gaacttctcc agccttgaaa attttagagc ctatgtggat ggattcgaac cgaacggctg 720
cattgagggc aagctttctc aaatgtcaaa ggaagtgaac gccagaattg agccatttat 780
gaagacgaca ccacgtcctc ttagactacc tgatgggcct ccctgctccc agcggtcgaa 840
gttcttactg atggatgccc ttaaactaag cattgaagat ccgagccatg agggggaagg 900
tatcccatta tatgatgcaa tcaaatgcat gaagacattt ttcggctgga aagagcccaa 960
tatagtaaaa ccacatgaca agggcataaa tcccaattac ctcctggctt ggaagcaggt 1020
gctggcagaa ctccaagaca ttgaaatgaa gataaaatcc caaaaacaaa gaacctgaag 1080
aaaacaagcc aattaaagtg ggcacttggt gagaatatgg cacctgaaaa agtagacttt 1140
gaggactgca aagatgttag tgatctaaag cagtatgaca gtgatgaacc agagcctaga 1200
tcgttatcga gctgggttca gagcgaattt aacaaggcat gcgaattgac agattcgagt 1260
tggattgaac ttgatgaaat aggagaagat gttgctccaa ttgagcacat tgcaagtatg 1320
agaagaaact atttcacagc ggaagtgtct cattgcaggg ctactgaata cataatgaag 1380
ggagtgtata taaatacagc cctattgaat gcatcctgtg cagccatgga tgacttccaa 1440
ttgattccaa tgataagcaa gtgcagaacc agagaaggaa gacggaagac aaatctgtat 1500
gggttcatta taaaaggaag atcccatttg aggaatgaca ccgatgtggt gaactttgtg 1560
agcatggaat tctctcttac tgacccgagg ctggaaccac acaagtgggt aaagtactgt 1620
gttcttgaaa taggagacat gctcttacgg acggcaatag gtcaagtgtc aagacccatg 1680
tttttgtatg tgagaaccaa tgggacttcc aagattaaga tgaaatgggg catggagatg 1740
aggagatgcc ttcttcaatc ccttcaacaa attgagagca tgattgaagc cgagtcctct 1800
gtcaaagaga aagacatgac caaagagttc tttgagacca aatcagaaac gtggccaatt 1860
ggggagtcac ctaagggggt ggaggaaagc tccattggga aggtgtgcag aacactacta 1920
gcaaaatctg tcttcaacag cctatatgct tctccacaac ttgaggggtt ttcagctgaa 1980
tcaagaaaat tgcttctcat tgttcaggca cttagggaca acctggaacc tgggaccttc 2040
gatcttgggg ggctatatga cgcaattgag gagtgcctga ttaatgatcc ctgggttttg 2100
cttaatgcat cttggttcaa ctccttcctc acacatgcac tgagatagtt gtagcaatgc 2160
tactatcggc ta 2172
<210> 31
<211> 2285
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 31
atggatgtca acccgacttt acttttcttg aaagtgccag tgcaaaatgc tataagtacc 60
acattccctt atactggaga ccctccatac agccatggaa caggaacagg gtacaccatg 120
gacacagtaa acagaacaca ccaatactca gaaaaggggg agtggacaac aaacacagag 180
accggggcac cccaactcaa cccgattgat ggaccattgc ctgaggataa cgagcccagt 240
gggtatgcgc aaacagattg tgtattagaa gcaatggctt tccttgaaga atcccaccca 300
ggaatctttg aaaattcgtg ccttgaaacg atggaaattg tccaacaaac gagagtggac 360
aaactgaccc aaggtcgcca gacttatgac tggacattga atagaaacca accggctgca 420
actgctttgg ccaacactat agaagtcttc agatcaaacg gtctgacagc gaatgagtcc 480
ggacgcctaa tagatttcct caaggatgtg atggaatcaa tggataaaga agtaatggaa 540
ataacaacac atttccagag aaagagaaga gtaagggaca acatgaccaa gaaaatggtg 600
acgcaaagaa caattgggaa gaagaagcaa aggctgaaca agaggagcta cctgataaga 660
gcgctgacac tgaacacaat gaccaaggat gcggaaagag gcaaattgaa gaggcgtgca 720
attgcaacac ccggaatgca aatcagagga tttgtatact tcgttgaaac actagcaagg 780
agtatctgtg agaaacttga gcaatctggg ctcccagtcg gagggaatga gaagaaagct 840
aagctggcaa acgtcgtgag gaagatgatg accaactcac aggatactga actctccttt 900
acaattactg gagacaatac aaaatggaat gagaatcaaa atcctaggat gtttctggca 960
atgataacgt acatcacaag gaaccagcca gaatggtttc gaaatgttct aagcattgcc 1020
cctataatgt tctcaaacaa aatggcgagg ctagggaaag gatacatgtt cgaaagtaag 1080
agcatgaagt tgcgaacaca aataccagca gaaatgcttg caaacattga ccttaaatac 1140
tttaatgaat cgacaaaaaa gaaaattgag aaaataagac ctctattaat agatggtaca 1200
gcctcattga gccctgggat gatgatgggc atgttcaaca tgctgagtac agtcctaggg 1260
gtttcaatcc taaatcttgg acagaaaaga tacaccaaaa ccacatattg gtgggacggg 1320
cttcaatcct ctgatgattt cgctctcatt ataaatgccc cgaatcatga aggaatacaa 1380
gcaggggtgg ataggtttta tagaacttgt aaactagttg ggatcaatat gagcaaaaag 1440
aagtcttaca taaatcggac tgggacattt gaatttacga gctttttcta tcgctatgga 1500
ttcgtagcca atttcagtat ggagctgccc agttttggag tgtctggaat taatgaatcg 1560
gccgacatga gcattggtgt tacagtgata aagaacaata tgataaacaa cgaccttggg 1620
ccagcaacag ctcagatggc tcttcagcta ttcatcaaag actacagata cacataccga 1680
tgccacagag gagatacgca aatccaaact aggagatcat tcgagctgaa gaagctatgg 1740
gaacaaaccc gttcaaaggc aggactgttg gtttcagatg gaggaccaaa cctatacaat 1800
atccgaaatc tccatattcc tgaggtctgc ttgaaatggg aattgatgga tgaagactac 1860
cagggtagac tgtgcaatcc tctgaatcca ttcgtcagcc atagggaaat tgaatctgtc 1920
aacaatgcta tagtaatgcc agctcatggc ccggccaagg gcatggaata tgatgccgta 1980
gcaaccacac attcatggat tcctaaaagg aatcgttcca ttcttaacac aagtcagaga 2040
gggattcttg aggatgaaca gatgtaccaa aagtgctgca atctattcga gaaattcttc 2100
cccagtagtt cgtatcggag accggttgga atttccagta tggtggaggc catggtttct 2160
cgggccagaa ttgacgcacg aattgatttc gagtctggaa ggattaagaa ggaagagttt 2220
gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa atagtgaatt 2280
tgctt 2285
<210> 32
<211> 2315
<212> DNA
<213>H5N6 subtype avian influenza virus GDJM plants
<400> 32
atgaacagaa taaaagaact aagagatcta atgtcacagt ctcgcacccg cgagatactg 60
acaaagacca ctgtggacca tatggccata atcaaaaaat acacatcagg aagacaggaa 120
aagaatcccg ctctcaggat gaaatggatg atggcaatga aatacccgat cacagctgac 180
aaaaagataa tggagatgat tcctgaacga aatgaacaag gtcaaactct ttggagcaag 240
acaaatgatg ccgggtcaga cagagtaatg gtatcacctc tggctgtgac ttggtggaac 300
aggaatgggc caacaacaag caccgtccat tatcccaagg tgtacaaaac ctactttgaa 360
aaggttgaaa gattaaaaca cggaaccttt gggcccgttc atttccggag tcaagtcaaa 420
atacgccgca gggttgacat aaatccaggc catgcggatc tcagtgcgaa agaagcacaa 480
gatgttataa tggaggttgt tttcccaaac gaggttggag cgaggatatt gacttcagag 540
tcacaaatga caataacaaa ggaaaagaaa gaagaactcc aggattgtaa gattgcgcca 600
ttgatggtgg catatatgtt ggaaagagaa ctggttcgca agacaagatt cctaccagtg 660
gctggcggga caagcagcgt gtatatagaa gttttacatt tgactcaagg aacttgctgg 720
gagcaaatgt acacaccagg aggagaagtg aaaaatgatg acattgacca gagtctaatc 780
attgctgcta gaaacattgt gagaagagca acagtgtcag cagacccatt agcatcactc 840
ttggagatgt gccacagtac acaaattggc gggataagaa tggtggacat tcttaggcaa 900
aatccaacag aagagcaagc tgtagacata tgcaaggcag caatgggtct gagaattagt 960
tcatccttca gttttggagg tttcactttc aaaagaacaa gtggttcttc tattaaaaga 1020
gaggaagaag tgcttacagg caacctccaa acattgaaaa taagagtgca tgaaggatat 1080
gaagaattca caatggttgg gcgaagagca acagcaattc tgaggaaagc aaccaggagg 1140
ctgattcaat tgatagtaag tgggagagac gaacaatcaa ttgctgaagc aatcattgta 1200
gcaatggtat tctcacaaga ggactgcatg ataaaggcag tccgaggtga tttgaacttt 1260
gttaacagag cgaaccaacg cctgaacccc atgcatcaac tcctgaggca cttccaaaag 1320
gacgcaaagg tactattcca gaactggggg cttgagccca tcgacaatgt aatggggatg 1380
gtgggaatat tgcctgatat gactcccagt acggaaatgt cattaagggg agtgagagtc 1440
agtaaaatgg gagtagacga atattctaac actgaaagag tggtcgtgag cattgatcgt 1500
ttcctgagag tacgggatca acaagggaac gtactcttat cccctgaaga agttagtgaa 1560
acacagggaa cggaaaagtt aacaataaca tattcttcat ctatgatgtg ggagatcaac 1620
ggcccagaat cagtgctggt caacacatac caatggatca ttaggaattg ggagaatgtg 1680
aagatccaat ggtcccaaga ccctactatg ttatacaata agatggaatt cgagcccttt 1740
cagtctttgg tacctaaagc tgttagaggt caatacagtg ggttcgtgag aacactattc 1800
cagcaaatgc gtgatgtgtt gggaacattt gacactgttc aaataataaa gctgctacca 1860
tttgcagcag caccaccaga gcaaagcaga atgcaatttt cttctctgac ggtgaatgta 1920
cgaggatctg gaatgagaat acttgtaaga ggcaactccc ctgtgtttaa ctataacaga 1980
tcaactaaga ggctcacagt cctcgggaaa gatgcgggtg cactgacaga agatccgaat 2040
gagggaacag caggagtaga gtctgcagta ctgagaggat ttctaattct aggcagagaa 2100
gacaagagat atggaccagc attaagcatt aacgagttga gcaatcttgc taaaggggag 2160
aaggctaatg tgttgatagg gcaaggagac gcggtgttgg taatgaaacg gaaacgggac 2220
tctagcatac ttactgacag ccagacagcg accaaaagaa ttcgtatggc catcaattag 2280
tgttgaattg tttaaaaacg accttgtttc tacta 2315
Claims (7)
1. a kind of universal primer group for expanding H5N6 subtype avian influenza virus full-length genome, nucleotide sequence are as follows:
Primer pair 1:
HA1-F:AGCARAAGCAGGG (SEQ ID NO:1)
HA1-R:TCTGCTCAATAGGTGTT (SEQ ID NO:2)
Primer pair 2:
HA2-F:CCAAAATRAACGGGCA (SEQ ID NO:3)
HA2-R:AGTAGAAACAAGGGTGR (SEQ ID NO:4)
Primer pair 3:
NA-F:AGGGTGAAAATGAAT (SEQ ID NO:5)
NA-R:CTCCTGTTGCTGAAATGCA (SEQ ID NO:6)
Primer pair 4:
M-F:TCTTCTAACCGAG (SEQ ID NO:7)
M-R:TTCTATGTTGACAAA (SEQ ID NO:8)
Primer pair 5:
NS-F:ACACTGTGTCAAG (SEQ ID NO:9)
NS-R:AAGCTGAAACGAGAAGG (SEQ ID NO:10)
Primer pair 6:
NP-F:CGATCTTATGAACAGA (SEQ ID NO:11)
NP-R:CTGCATTGTCTCCGAAG (SEQ ID NO:12)
Primer pair 7:
PA1-F:AGCRAAAGCAGGTACTGATY (SEQ ID NO:13)
PA1-R:CATNCCCATCATCAT (SEQ ID NO:14)
Primer pair 8:
PA2-F:ACATTTTTCGGCTGGAAA (SEQ ID NO:15)
PA2-R:GAAACAAGGCATTTT (SEQ ID NO:16)
Primer pair 9:
PB11-F:GAGACTGGAGCACCYCAACT (SEQ ID NO:17)
PB11-R:AGCRAAAGCAGGCAAACY (SEQ ID NO:18)
Primer pair 10:
PB12-F:CRAAAGCAGGTCAAWTATAT (SEQ ID NO:19)
PB12-R:ACAAGGTCGTTTTWAAACW (SEQ ID NO:20)
Primer pair 11:
PB21-F:ARTTCACAATGGTTGG (SEQ ID NO:21)
PB21-R:ACAAGGCATTTTTTCA (SEQ ID NO:22)
Primer pair 12:
PB22-F:TAATAAAGCTGCTACC (SEQ ID NO:23)
PB22-R:CAGCAGGTYCCTT (SEQ ID NO:24).
2. a kind of kit for expanding H5N6 subtype avian influenza virus full-length genome, it is characterised in that: the kit, which contains, has the right
It is required that primer sets described in 1.
3. kit according to claim 2, it is characterised in that: the kit also contains dNTP, Taq enzyme, reverse transcriptase.
4. a kind of method for expanding H5N6 subtype avian influenza virus full-length genome, it is characterised in that: the following steps are included:
1) sample rna is extracted, is cDNA by RNA reverse transcription；
2) using cDNA as template, 12 pairs of primer pairs described in claim 1 carry out RT-PCR amplification respectively, obtain H5N6 hypotype
The amplified production of avian influenza virus full-length genome.
5. according to the method described in claim 4, it is characterized by: RT-PCR amplification system is in step 2)
6. according to the method described in claim 4, it is characterized by: RT-PCR amplification system program in step 2) are as follows: 95 DEG C pre-
It is denaturalized 5min, 95 DEG C of denaturation 50S, 58 DEG C of annealing 30S, 72 DEG C of extension 3min, carries out 35 circulations, 72 DEG C of extension 5min.
7. a kind of sequencing approach of H5N6 subtype avian influenza virus full-length genome, it is characterised in that: according to claim 4~6 times
Method described in one obtains the amplified production of H5N6 subtype avian influenza virus full-length genome, by the full-automatic sequence of amplified production
Column sequenator is sequenced and is spliced to get the nucleotide sequence of H5N6 subtype avian influenza virus full-length genome.
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