CN107245533B - A kind of crucian coronavirus TaqMan real-time fluorescence quantitative RT-PCRs detection primer and application - Google Patents

Abstract

The invention belongs to field of virus detection, specifically disclose a kind of crucian coronavirus TaqMan real time fluorescent quantitatives RT PCR detection primers and probe and application, and inventor has isolated a kind of crucian coronavirus from crucian for the first time, which is crucian coronavirus（Crucian Carp Bafinivirus）HB93 designs primer and probe for the virus sequence：Forward primer F：5'ATCAAACACCCACTCCCAC 3', reverse primer R：5'‑AGGTCAGGTGATGTTGCG‑3'；Fluorescence probe is P：5'‑FAM‑TGACCCATTCTTGCACTGCTTCTGT‑TAMRA‑3'.Quantitative detection, specific good, the high sensitivity of the primer can be carried out to crucian coronavirus.

Description

A kind of crucian coronavirus TaqMan real-time fluorescence quantitative RT-PCRs detection primer and Using

Technical field

The invention belongs to viruses molecule detection technique fields, and in particular to a kind of crucian coronavirus TaqMan real-time fluorescences Quantitative RT-PCR detection primer and application.

Technical background

International Commission on Virus Classification (International Committee on Taxonomy of Viruses, ICTV the 9th report of newest virus taxis) has been delivered in 2012, it will be coronal in shell type virales (Nidovirales) Coronavirus subfamily (Coronavirinea) and Torovirus subfamily are newly set in Viraceae (Coronavi ridea) (Torovirinae)。

Coronaviridae is a kind of linear single strand plus RNA virus, is most separated earlier than nineteen thirty-seven from chicken, Alme Ida etc. has carried out these viruses Senile Mouse, and electron microscope observation finds there is likeness in form corona on these viral coatings Spinous process, therefore propose name this viroid be coronavirus^[1].1975, international viral nomenclature and classification committee definite designation Coronaviridae.During 2002-2003, from the atypical pneumonia of China some areas generation, i.e., by coronavirus genus SARS virus (SARS-CoV) causes, and has a countries and regions more than 30 to report appearance disease in succession in the world Example, case fatality rate are more than 10%.2012, Middle East started novel coronavirus occur, and case fatality rate is more than 50%, causes generation The extensive concern of boundary's scope causes fear of the society to coronavirus.Therefore, further investigation is carried out to coronavirus with important Meaning.

Coronavirus subfamily is concentrated mainly on to the research of coronaviridae at present, and to the research of Torovirus subfamily very It is few.Wherein, the bream viral (White bream virus) in 2006 bream Tobamovirus (Bafinivirus) is by Sch ü The scientists such as tze H have found in bream body, this be also determined in current ICTV classification it is unique using aquatic animal as host's Coronavirus.

At present crucian coronavirus (Crucian Carp Bafinivirus, CCBV) there is no it is open report, more also without having The detection method of effect.

The content of the invention

It is an object of the invention to provide a kind of detections of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCRs Primer, the primer are as follows：

Forward primer F：5'-ATCAAACACCCACTCCCAC-3', reverse primer R：5'-AGGTCAGGTGATG TTGCG- 3'；Fluorescence probe P：5'-FAM-TGACCCATTCTTGCACTGCTTCTGT-TAMRA-3'.

It is an object of the invention to provide a kind of detections of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCRs The application of primer is prepared into crucian coronavirus detection kit using the application.

Final object of the present invention is the provision of a kind of crucian coronavirus HB93TaqMan real time fluorescent quantitatives The positive control of RT-PCR, the positive control are that sequence shown in SEQ ID NO.2 is inserted into pGEM-T carriers to obtain.For Reach above-mentioned purpose, the present invention takes following technical measures：

A kind of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCR detection primers, the primer are as follows： Forward primer F：5'-ATCAAACACCCACTCCCAC-3', reverse primer R：5'-AGGTCAGGTGATGTTG CG-3'；Fluorescence Probe P：5'-FAM-TGACCCATTCTTGCACTGCTTCTGT-TAMRA-3'.

A kind of application of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCR detection primers, using upper It states primer and is prepared into the detection for detecting crucian coronavirus detection kit or being directly used in crucian coronavirus.

Preferably, when being prepared into kit, the nondiagnostic detection method of the kit includes：

A kind of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCR detection methods, step include：

1) it is cDNA to extract the RNA of sample to be tested and reverse transcription；

2) PCR system

Fluorescent quantitation reaction solution by 10 × Taq reaction buffers, 2 μ L, 0.4 μ L of 2.5mmol/L dNTPs, 50 μm of ol/L just To each 0.4 μ L of primer and reverse primer, 25 μm of 0.4 μ L of ol/L probes, 0.4 μ L of 5U/ μ L Taq archaeal dna polymerases, aseptic double-distilled water 14 μ l are formed.Cycling condition is：95 DEG C of pre- change 10min；94 DEG C of 10s, 60 DEG C of 45s expand 40 Xun Huans.The journey of fluoroscopic examination Sequence carries out at the end of being arranged on the second step of each cycling, Detection wavelength 530nm.

3) testing result judges,

Ct value≤30, and there is typical amplification curve, then result is the positive.No Ct values or without amplification curve, then result For feminine gender.Ct values>When 30, the sample is reformed, is otherwise the positive if it is feminine gender to reform result without Ct values.

In the method, it is preferred that positive control is that sequence shown in SEQ ID NO.2 is inserted into pGEM-T carriers to obtain Compared with prior art, the present invention has the following advantages：

1st, high sensitivity, in 50-5 × 10⁹Copy number scope quantification is accurate.

2nd, detection speed is fast, only 1 it is small when, in addition prepared by RNA extractions and reverse transcription, altogether when only needing 3-4 small；

3rd, it is easy to implement the method, it is easy to operate；

4th, high-throughput sample detection can be carried out at the same time.

In order to realize above-mentioned purpose, the present invention uses following technical measures：

Specific embodiment

Technical solution of the present invention is the conventional scheme of this field, agents useful for same or raw material if not otherwise specified, If not otherwise specified, it is purchased from commercial channel or disclosed.

Embodiment 1：

A kind of crucian coronavirus HB93 (present invention or be CCBV-HB93), separation process is as follows：

1) will take after the tissues such as viruliferous crucian liver, brain, spleen, kidney take out and mill, culture medium dilutes 100 times, After 2000rpm centrifugations supernatant is taken to filter, it is single to be inoculated into grass carp gonadal cell (Grass carp ovary cell line, GCO) Layer, 25 DEG C are cultivated 7 days, generate cytopathic effect (CPE), it is seen that GCO cellular contractions are rounded, diopter increases, part cell It starts shedding off, cell monolayer starts to rupture.

Culture medium：M199,10% hyclone, pH 7.0~7.2

2) sick cell is collected, Morphological Identification is carried out using transmission electron microscope.

3) virus is collected, genome sequencing is carried out to it, sequence is shown in SEQ ID NO.1.

The morphological feature of crucian coronavirus：For virus in rodlike, surrounding has apparent corona shape spinous process structure, and typical Coronavirus form is consistent, which has sent to China typical culture collection center on May 11st, 2017 and carried out preservation, point Class is named：Crucian coronavirus (Crucian Carp Bafinivirus) HB93, deposit number：CCTCC NO：V201722, Address：Wuhan, China Wuhan University.

Embodiment 2：

A kind of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCR detection primers, the primer are as follows： Forward primer F：5'-ATCAAACACCCACTCCCAC-3',

Reverse primer R：5'-AGGTCAGGTGATGTTGCG-3'；

Fluorescence probe P：5'-FAM-TGACCCATTCTTGCACTGCTTCTGT-TAMRA-3'.

Embodiment 3：

A kind of crucian coronavirus HB93TaqMan real-time fluorescence quantitative RT-PCR detection methods, step include：

1) it is cDNA to extract the RNA of sample to be tested and reverse transcription；

2) PCR system

Fluorescent quantitation reaction solution by 10 × Taq reaction buffers, 2 μ L, 0.4 μ L of 2.5mmol/L dNTPs, 50 μm of ol/L just To each 0.4 μ L of primer and reverse primer, 25 μm of 0.4 μ L of ol/L probes, 0.4 μ L of 5U/ μ L Taq archaeal dna polymerases, aseptic double-distilled water 14 μ l are formed.Cycling condition is：95 DEG C of pre- change 10min；94 DEG C of 10s, 60 DEG C of 45s expand 40 Xun Huans.The journey of fluoroscopic examination Sequence carries out at the end of being arranged on the second step of each cycling, Detection wavelength 530nm.

3) testing result judges,

Ct value≤30, and there is typical amplification curve, then result is the positive.No Ct values or without amplification curve, then result For feminine gender.Ct values>When 30, the sample is reformed, is otherwise the positive if it is feminine gender to reform result without Ct values.

Standard positive template is connected containing 389 nucleotide fragments of crucian coronavirus gene (shown in SEQ ID NO.2) Access what pGEM-T carriers were formed.And the carrier can be proliferated in E. coli DH5a.The plasmid converts Escherichia coli With alkaline lysis method of extracting after E.coli DH5a multiplication, through DNA Purification Kits, A is measured with spectrophotometric₂₆₀It is quantitative simultaneously It is diluted to 1 × 10⁹Copy/microlitre, -20 DEG C of preservations.

Embodiment 4：

The foundation of the quantitative equation of kit prepared by crucian coronavirus fluorescence quantitative RT-PCR primer and sensitivity examination It tests.

A) prepared by templates:

It will connect (shown in SEQ ID NO.2) containing 389 nucleotide fragments of crucian coronavirus gene and be carried into pGEM-T With alkaline lysis method of extracting after the plasmid conversion bacillus coli DH 5 alpha multiplication of body, through DNA Purification Kits, spectrophotometer is used It is that 200ng/ μ L, A260/A280 value are 1.81 to measure positive recombinant plasmid concentration.

And the DNA copy number of recombinant plasmid is calculated according to following methods.

Molecule copy number (copies/ μ L)=6.02 × 10²³× DNA mass concentrations/DNA molecular amount, wherein：DNA mass Concentration=260nm absorbance × 50 × extension rate；DNA molecular amount=DNA base number × 324.5.

B) prepared by standard curves

Recombinant plasmid is subjected to 10 times of gradient dilutions, with 1 × 10⁹、1×10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、 1×10³、1×10², 10copies/ μ L as standard items template, establish standard curve.The restructuring of different dilution gradients is taken respectively It is each to sequentially add 10 × Taq reaction buffers, 2 μ L, 0.4 μ L of 2.5mmol/L dNTPs, 50 μm of ol/L primers Fs/R by 5 μ L of plasmid 0.4μL、

25 μm of 0.4 μ L of ol/L probes P, the 0.4 μ L moisturizings to 20 μ L of 5U/ μ L Taq archaeal dna polymerases are by above-mentioned reaction system PCR amplification, response parameter are carried out after mixing：95℃10min；95 DEG C of 10s, 64 DEG C of 45s, totally 40 each dilution factors of cycle set 3 A repetition.The visible apparent amplification curve of 50 copy number recombinant plasmids.According to plasmid copy number and the correlation of Ct values, analysis is marked Directrix curve.Copy number (x) and the relation of Ct are：Ct=-2.773lgx+31.577, coefficient R²=0.9904.

Embodiment 5：

Crucian coronavirus fluorescence quantitative RT-PCR detecting kit specific test

Using the method described in embodiment 1, the viral sample to be checked in table 1 is detected, while negative control is set Group (distilled water) and positive controls (CCBV-HB93), viral sample to be checked are specially：Huichun viremia virus (SVCV), Grass carp reovirus 873 (GCRV-873), grass carp reovirus 9014 (GCRV-9014), infectious hematopoietic organ necrosis Viral (IHNV).

1 crucian coronavirus RT-PCR detection kit specific test of table

The results are shown in Table 1, and only the visible typical amplification curve of positive controls (CCBV-HB93), ct values are：21 illustrate crucian carp Fish coronavirus HB93RT-PCR detection primers possess very strong specificity.

Embodiment 6：

With the clinical mark of PCR kit for fluorescence quantitative detection crucian coronavirus of crucian coronavirus Quantitative detection This：Illness crucian acquisition tissue sample doubtful to 3.

As a result it is as follows：

3 parts of samples altogether are all positive, positive rate 100%.The Ct values respectively 22.5 of doubtful illness crucian tissue, 23 and 25.The coronavirus amount that doubtful illness crucian in reaction system is calculated according to standard curve is respectively 10^3.27Copy number/ μL、10^3.09Copy number/μ L and 10^2.37Copy number/μ L.

It can illustrate from above-mentioned experiment, fluorescence quantifying PCR method quantitative repeatability is good, quantitative accurate, moreover, fluorescent quantitation PCR detection kit only needs the detection of sample 4 hours can just complete, and traditional PCR methods about need 7 hours or so ability It completes, therefore, detection time can be greatly shortened using the kit.

The operation of the kit only needs 1 people that entire quantitative work process can be completed, once 32-384 detectable (by The model of instrument determines) sample, this also reduces the wastes of human resources.

SEQUENCE LISTING

<110>Hubei Import And Export Inspection And Quarantine Technology Center

<120>A kind of crucian coronavirus TaqMan real-time fluorescence quantitative RT-PCRs detection primer and application

<130>A kind of crucian coronavirus TaqMan real-time fluorescence quantitative RT-PCRs detection primer and application

<160> 5

<170> PatentIn version 3.1

<210> 1

<211> 26000

<212> DNA

<213>Artificial sequence

<400> 1

ttgaattacc aataaactac cacccgctat catagcacta gacactagcg aactgctagc 60

ttcctccaag ctgcacaacg caaactgtct ttttaactct cctaacctcg aacgcagcgc 120

aggtaccctc acccggacca cgcgtgtgcc cttgactgga aaccctacct tcgatcaggc 180

cttcgaacgc agcaaaggta ccctcacccg gaccacttgt gtgcccctac gcctgctctt 240

acgtagcccc atcctaccca cccgccaact cttaacctat cagctttgcc caccgtgacg 300

actagacgtg atctaactgc tactaccaat cctattacaa ccaacttgcc caccaagacg 360

actagagagt gacgtaactg ctactgccta aggcactacc agcgaaatca taatctgctc 420

ctcttgaccc aagacggttt ggaacggtgc tactagtgaa ctcacttctg ctccttgcga 480

cccaaagacg gttattattt ggaaaacctc ttacagacta gagtcttgaa tctccactgc 540

tactgtcgaa gtcggtgtgc tgcctttata aagccggtga actgctactg ttcttagaac 600

attatcagga atcctgctga attttgtcag ctatccctcc aagcgctact agtggtctct 660

tacctctact ctgccccttt acctactcaa tataacttaa atctcttcta gaatcgcgct 720

agcgcaaacc ttcaccggta accgccagaa cgtcctacaa gctctcaacc accggcactt 780

tgttgctaaa tcctacgtgc aagcatcttt ttgctataac atccacgttc gaaaatcaac 840

aaaaatgccc aacatgcgaa tccgcaagat tgctcctaaa gccgctgtta aaccaaccca 900

tagcggccca agagtggtct ccaatcccgc aagctgccac atttacatcg gaccccaact 960

gcgtgctcac aactacccga gcgtgctaaa gggttccttt aaggctgcca gcgcggaaac 1020

cataacagct atcaacctaa ttgccaccgt ccgcaaggat gctggtattt caggtcaata 1080

tgctgggaag atgggttatg accccgagat gggacttttc gtttccgtcg aaggtgttcg 1140

tgctctccaa gcttccaaac tccaaactgc ccttaaggca attaaaaagg gtactagacc 1200

ctttggcctt ttcaggccta aacagagtgt gactaacacc cacgctcaaa atttgctagc 1260

cctccaccgt gctagctcca ccggcccttg gaccaaacac ttaccatctc aaggtcccat 1320

aggtaactgc ctcgcagaat tcatgcagga gtacactacc acaattccac catttgatag 1380

tgtcctcact gaaaacggtt ggcaatacgc tgtcacgatt gacggtgtga ctaaatctcg 1440

cctcccgtac accgaaattc gccgcggtgg ttatgaattt aaaacaacta attaccacat 1500

cggtgcaatc caggttactg ccaaccacat tggcaaagta aaccctggag atactaaatt 1560

ggactgtcaa tatttcatgt tgacacgtaa tcaaaagccc caaaacttca ctgctgagca 1620

actcgaagga aacatccata acatcatctt cgacctcagt catgaaactg gcaaacccaa 1680

actctatgga atggaagaaa cacgacaatg ggctgggaca acaccaaaac cacctaagaa 1740

aaagcttaga cttgtagcta tgacatcaac tctcagtgcc actccatcaa ccacacaccc 1800

agcgccccta acaaccacac gcgcaccact taaaacacac atttcaattg gcccgcacaa 1860

ctgcaccatc atagatatta ataaagccca attcgccttt gataactaca cttctcaact 1920

ccctaactct tgtgttcacc gtgccaacaa agctgtttgc gccggtttga atggctgatc 1980

ctttcctttt cctacccact ttaactacct ctctaaagta gaaacatcta aaggaactta 2040

tcaattgggg tacattttag ccccaaaact acggcccctg ctatccaatt ctacgtcttg 2100

caatttcaga accacgtcta cctacgtctt ctccccatgc ctatgaacct ttctttccgc 2160

actatctgcg acattgaaac cgcccacggt caaccacacc caacttacac aagcccagac 2220

tggcgcgtgt attacctcgc aaagtacgat cccaccaaag tactcagagt tcaagttgtt 2280

gattgggcag gttcagatac cattactgaa gatcatgtca ccctcttccc acgccgacac 2340

attatgacta aagttttgaa aaacgaccaa actaaagtta actacatcga cccaactaaa 2400

gatgcagcaa caacaccctt cacaattgaa acagttgaag gaggcaaagt tgaagtactc 2460

tccgccaacg tcatccgtca aattaacatt gaaactggtc gcggcaagaa acgtaaaaca 2520

gctaaattag atgtaaaaat ttcaattaga gttaaagcta ctaaaacccg cacaacaatt 2580

cacgtcttca taacccaagc aaaagatgaa agcaaaatcg tcactccaac ctcagaaatg 2640

cagggagtag ttgacgtttt aatttccaga gcatgtgtcc tctcacaaca cgcaggacct 2700

gacatgactt atgaactgca cacacctgac ggttcttgca ctaaccccct cgttcaattc 2760

tttgaagaag aaggcgaggt tcctgaagat gtttacgatc tcttcccatc tgtagaacgt 2820

aacgctgaag gcttagttat gtgcatgcca gaaggttttg ccctcatctc tggttgcatt 2880

cctcatccac ttgcttacta tgctgacaag tacggtcaaa cactcgtacc tgaattcaaa 2940

gtgacaagaa ctcacgttga acgcggcccc attccaccat cacggtccat ttactacaca 3000

ttcatcccaa ttaatgtaga ctccatccca cccttcggac acacctacaa atccctcgcc 3060

aaacttaaca catttaacct gggttgggaa gatcaagaca tgttagataa cgctgacacc 3120

ctccttccat gtctcttaga aaatgaaaac atgaaacttc acctaacatg gaaaatcatc 3180

gcccgtgact tctcagataa caccactgaa acagccctta ctacctacac tgattttaca 3240

actagtgatt tcaccccagc caaccaaaca ggatccagct ctcaatccct tagctctcca 3300

cctctttccc aactctcttc ccacaaatct cttgctaacc tctcagaaac tggctcaata 3360

gataacatcc cggaaaacac ccctgaaaca atccaagaag aagaagatat cgaagaggaa 3420

gtaacatctc ctgttcaacc cacactcaac acactccaag aacaagctac tctcaacgaa 3480

tccattcaat cactcgttga atcagttgat tctgatgtcc cttacacctg ggaacaacgt 3540

gttgaagaaa gtgaggttgc aactattgat ttacaagctg tagaattaga tcaaacaccc 3600

actcccacca ctctctcaac agaagcagtg caagaatggg tcaacgcaac atcacctgac 3660

cttgctacag tagactggaa cagtgcaata actgaacagg aaaccacagt aacttcacta 3720

atagaagaaa tctccctacg ccccaacacc aacaacaccg gctataactt gtttaaacct 3780

aaccaacgta acaacaacag cccatcagac tacctatcac gaacaacatc agcaaccaaa 3840

cactgcttaa ttaacggtct tacttcaggt tccaccatcc ttgaagtcgg atgcggatat 3900

ggtaaagaat tccacaaata caagaactct aacatcaaag tcgttgcttt ggaacctcac 3960

catgaacgct acaccgaagc cgcgaagaaa ctaccaccaa attctaccat ttacaacaag 4020

accctagaag aatacaacca aattgtcatt aaagaacaaa ttgacgccct agtttccttt 4080

aacgccattc attacagtac agttactgaa gctcttaaag catgtaacct tgccaaagat 4140

caagttgtca ttttaacacc aaatcatgat aaacttaaag ctcttcacga tagtaacaca 4200

accatcacaa attacaaaat tgaacttgct aataacaaat ttacatttac aatcggccaa 4260

aacacctaca gcgaaaacta cttctcactt caggatttca tgattggaat gcatgccagc 4320

aacttcatgc tttcttcccg ctcttgtact ctgcttgaac atgttaacac cttcaatgcc 4380

cacctcttaa ttgacgccac aacagaaaca actaccctgc tagacgccta tgaagtactc 4440

gtcttcataa aatttagacc actgctcctc acctactcca acaccgctct tgcaactaac 4500

aactcaagaa caatccgcag tgcactctca cgtcttatta acttccgtgt cgcaccttct 4560

aaaacaacat cacaggtaca tgttcacatc ctcacccgca acgaccacgc aaccacagaa 4620

caagaacatc tagacttagc aaatattgaa attgcccaca tgaacccaaa accagtaatt 4680

atctttgtaa cttcttcccg atccaaacat aactacacca ccccaactgt cttcttcgac 4740

tttgctacac tatcagcccc caccctccca gaacagctta aaccactcac tgatctagcc 4800

ctcgcaatcc aacacactgc taccaaaact ccaacaacag tacttacaac tgccccagag 4860

gacccaaaaa ccctcccagc agcacccacc tcctttacaa aaccttcccg taaacgctac 4920

cactgcttcc acgatttaac caccctacca gttaacaaaa atttcatctt agttaacgca 4980

ggtaaccccc acttaaaatc aggtggtgga ataactaaag ccgtctataa tcgctacggc 5040

caaggattca tgcaaactaa caacagcatg catgctacac aattacctct tgaagttggt 5100

gacgtcttgg aatacccctc tgaccgtaac aacctgatta acgtgacagc cccctgcatc 5160

aacatcaaac acgcagatta caaaggccca caaccagccc acgcactact agcaacatat 5220

aacgcaatta tcaactacgt tgaacgtaag tacgataatg gtgacgagaa acacgtcttc 5280

attcctctac ttggtgctgg atactacggt tggtcacaga tccaaagcct tcaagctttt 5340

ctatccgcga ctgataatag tttccgcaac acatatgttc tactcctaca cccatcatct 5400

gtagatgttg aaacagctga agtatttgca aaaatgctcg ccactactaa cccacactac 5460

taccatgttg agggcatagc taaagttaat gccaactctt gcgttctgcg tgcccacatt 5520

gaccttgccc gcaaaacatc ttaccctcaa cctagcttag gattaatcgg cgcttactgg 5580

atgtacacct gccccaccct ggccgcccca ttgatacaag ctggcatgca cggtcccaac 5640

acctggactc ctggcagctt tattgatact agtattgctg aaatgactgc tcgcgcttac 5700

aaaataaatc ttgtttgcga cggttgcttc actgatatca tcaatttcaa tcccgaccaa 5760

ggtttaatct gcaatacctg taagacacct gtcactctcc ggacaaacta ttatcgccca 5820

cgcctcccac acatgattaa aaatagtcac ggacaaccca tccctaacat tattctggat 5880

ggaatctacc acatggccac gcaccaattg ttcctaaatt cggtactctt taaaaattca 5940

catgctttca caattaacaa cctaagtgaa tgtgatctcc caacaactac agcagtagca 6000

acctactttt acaaaccttc agatgcttta aacttcttcc tgaactttaa ctctgctatt 6060

tctgctaacg taacttatac atcagaacag ataacacaag ctcaaaccac ccctcaactc 6120

accactgatg atgactactc ggacttggac attgacacaa ttaaccctac acctactact 6180

ttaccacgcg tcccaacacc gctttcaatt agtgatagta gcgaagatat ctcactctcc 6240

gatgacaccc acaccgtagc atcccatcac gcttcacaaa tgtccataac agccgacata 6300

actagtgata gatatagctc tacaaactct ctagctatct cagaagatat aacatttcaa 6360

acaggtgacg gtgaaacaac aacttcactt ctgactgatt cggtcctata ctactctgtc 6420

gttacagtct tacaactacg taaagcagag gattgtgtag aacctgctgt agaaactatt 6480

aacctaagac ccaactcatc acttcatacc ttgacacatg acacaaacgc catcgaccaa 6540

ctttacatcc aagtggcttc tgatgcttca aatcacactg taatcaataa caccacactt 6600

gaatctattt ctaaaacgat gtgtgtgaaa aactccttgt acacccttct tactaactca 6660

aagtgtataa gtcaccacat catagttaca ggatatgacc aacatgctca tgaaacacat 6720

accattaaaa gcgtcacgcg ccaaactatg ctcgccatcc aacctctcgt acaactcacg 6780

cccacacacg tttacagcac acttgaagaa gatatttcca ccatctttca aactgtcgtc 6840

ctagaacatg tcaaccacac taagctttcc acttataaca ccaagctaca ctacgcaact 6900

acagtaccaa caaacctact catcgatgga acaatcgaag atgttcaagg taagctgcgc 6960

gaagttgatt tcggcattgt tgttccttac cagtgtgaag ttgacgagca aattgaatac 7020

acaaatgcaa ttggcgactt actctaccaa aacacaaatc accactcacc actagtgacc 7080

tttgtttcta tcgacaaatc ttttactaaa cgtattttta attctttaaa gtttcaaact 7140

ctacagaacc tatggctcta ccttattacc ctacaagtat ttttcacagt taaaggaatt 7200

ggcttcggct tcagctcaac ggaaccaaac acacaactca tgaacggtct ctggatacaa 7260

tacggcatct tctacttcca agtacacctc caaaccctca tcacccaccc agtgaaaaat 7320

ggctacagac tactttcatt accaatcatg ctatacaaca aaggcaaaag attcaccgaa 7380

cgttcagtcc aaacaatgac gggagaaatc gttaacatac gaaaaccctc gccatcaaac 7440

gctgtacaaa aatacggcat taacacccga cttgaatacg ttgacatctt cgatctcctc 7500

cactatacac ctggatacat cgcatttaat ttcgccttgt tgtacctttt ttctggtaat 7560

ggttacataa cattcttagc ctacaccatt caattcttca ctgctcgtga catttacaga 7620

ctctctaaac acctttacac agtctcaaaa ctcactacct tcctctcgaa attcaaaaca 7680

gcaaccaccc gccacgtctt gtacgcaact aatcaagtaa ataaccgttc cctccacctt 7740

acccttggat ccacaacagt tgacttactt agctttacaa gcaaagcagt ttttctcgcc 7800

ctgtgctctt ctttcatttc atggcatttc ggattagctc ttgccggcgg ccactctggt 7860

gaagactact ctcgaccacc ctaccatgca atatttcaac acgcactccg cactttcgga 7920

cttatccctc ctaaagaaca ttactatagc tacccttccc tagcttcagc ttgcgcagac 7980

tcctcagctc cactttgctt cctgggaaca ccatttaact ttcacttccc atcatcctac 8040

acacaagcag catccatgac tactgaacac gtagtaccat tttggattca actaatcatc 8100

ctagccccac catcgctttt cctagttgtc atcccgtggc tgatcacaat gttcatctca 8160

ccagtattaa catttaacca acttttgctc cctgcactcg ccgtacattt cttagctatc 8220

tatgtaagtt acctttacta ttttaagaaa tcatgctgtc attctcacac atgccttaaa 8280

cacgctaaat tagaccgccc catccaaatt aaaggcctct ttgatttatc catgtacttc 8340

aatcacaata aactttgccc ccgccacaac ttctactgta ataatgctga cgaacccggc 8400

cacacacttg gacccgtcct agcagctcac atcactgaat tttaccatct taaacctggc 8460

actattaagc ccgatgtcgc cctagtacac acagaacacc acgcttctct gagtcgcaaa 8520

gaattgaaga aacacgacga cccaaccgat tgtgtctgga ccgccccaca aggcgctgct 8580

gatactctcg tctacgaagc ttacgctcat ttgaaaggag tccaaattcg cattgatgcc 8640

aacaccgata caacaactcg tagcatgcct attcccatgg gcgctaagac aacatcttct 8700

ccagctacag gtgttatcaa cacaaaagtt ctcggccatc ttaaaagaca ttgtaaaaac 8760

ccggctctcc aatcttacct atcaggatac gtcggcgccc ctaacgccaa aaccactatc 8820

atgcccatta cgctctttaa acatttgcct gaaggcctta agaaatacgc catcaaaaac 8880

atcaccttct caattaacat gctaaccgat aactctaact caggtttcat tccagaaggc 8940

atacacaacc caaacaacct cccaaccaag actttcctgg tcaaatctat tgatcaaaca 9000

atctttgacc aactccaccc tgaaacacaa gctatgctta aagacgaata ccacattaaa 9060

cttcatgaca taacatctca attttacgaa gttatttctt caatgatgtc ctacgaaagt 9120

ttcatcttcg cactcatcat cgtcttatcc gtctttctcg gtttcatctc ctacagctct 9180

accattactg ccaccacatc cctacatgcc ggcttgaacc ccaccatgcg cggatccacc 9240

acaggtaaac cattcatcca accagttact taccccaact ccattctaat ccctctgaca 9300

tcttcccaat ataatgtatt cagactacaa aacggaactc tctacttcac ttctcaaata 9360

actgctccaa cctgtaccgt taaactacca aacccctaca ttggcttaac cacagaaaaa 9420

ttctatgaat gctcatctgc cctcctccac atgcccttca tggttcatct tggcggtgtg 9480

aaaattgctt accttagagc cggtgttgcc tataacatgc caacatactc agagacacca 9540

caaactagta aactctgtgt tctcactgca ctctcaaaga cgaaatgctt taacatccta 9600

ccaggcggtt tccacacccc agtgctagcc atcctcgttt caacttgtgc acttctggca 9660

acggtcctct tctaccttta cctactccag atctttaaat tctacaccaa ctcagtcttg 9720

atggtggttg ccattcaaat tgcaactatc ttcctcttca tgctatcccc agctcttgct 9780

ctaacacttc aactggtctg tctcatgtac ctttaccata actggatggt tgccatgatt 9840

aacatcgcaa acctcgcagt tctagcccac acaacatttg gcatggtact catcttcatc 9900

tacgcgctct acagacttta caaagtctac caagttacat caaccgctgc agttacaatt 9960

aacgcaactg gtgaaatgcg cttccacgga actcttgatg aagttgctct ctgcaccttt 10020

ccaattagct ctgcaagtgt ttaccaaatc atgtctgacc tcagcttaga ttacattaaa 10080

ctggccagct acaaaacatc ttataaccgt gctacccgta acctggttaa catgatgcaa 10140

agatatattc tcgatacctc catcccagct atactctatg acggctcagt aacaactggc 10200

atcatgcaac aatccctagt tagacttaaa tccctagtta caacagttta caaggataac 10260

tactccaaca tctgcaaaat tttatcaaca acccactcac gatcaacatt ctgcgtcgga 10320

actttcctta acccaaccac tgttatcact tgctcccacg gaatcttagc aaactctact 10380

attaccgtct cccacaagtc aactatttat gaagcaacaa ttgaatcaca acgcaatgat 10440

atcacaattc ttaaactcac aacaccaaac tccgcaatca ctcacaccat cgagtgccat 10500

tcaccaaatg acgctgatct cgaaaatatg caaattaact acacccaatt tgttactttc 10560

gctgataaag atcactcaga tgccatagcc atgaacaaca caacctattt accatctggt 10620

cacttctttg ctatcggaac tgaatctggc gaatctggca gtccctactt ctttaacaac 10680

aaacttgtag gaattcacta cggagttaac acctgtgatc aatttatgct cgcctctaaa 10740

cctgatggca gttactacat cccagagatc caacacaaca tctccacaca accatctttt 10800

agccttagtt gcatagccca tggtcgaaaa tcggtaaccg ccaaaaacac ccccacactt 10860

cacgatgcta tcaccaaagc ccttgcccaa acaacacgct cagcttacac caacgattta 10920

acaatagacc acacacctga acaacttcag gctgtccttg actttctgac aaaccctctg 10980

aataaattag gccctaagac actccacgac tacatggcaa catccatcca agttcaatct 11040

accaccacag cccaatccgc ccacattgac gtccacaaac caattagaca agcagcaact 11100

ctctactatc taatggttat catttttgac atcatcgcat tcttcttaac ccccggttct 11160

gatttctcta atcaacttgt atccatgact gcctacctag ctgcctggtt ccttaataaa 11220

acatgctttt acaacaccca tgtcctttac acagtaacca catgcttcca actctgcaaa 11280

tacttcaaca tcgtctatct tttcacaatc attcaatcct ataaaccaat tgaaatgtca 11340

atgatggata aacaatatga tttcctttgc gccatcattg tagccctctt actctctcca 11400

atcctggtcc tcatctggaa acgcctctgc aacttcacct ggcattatct tgtcgctcat 11460

ctagttgttg gttgtgtgct ttgcatccac gtcttcttca gctacagatc ctttacatct 11520

tcaacaggcg gtctattaac attttcagaa ttcgccttta actcaccatt aacagcagtc 11580

ataacagcta cagttgatga aagtcctgtt tctgttattg tctacccact cttgcagcat 11640

gtcatcagcg caaccctgat cttagcaatc gtgccatctc ccctcctcgc agcaacagtc 11700

tactctttct atctcattga ccaagatgtc ctctgcctcc taatctttct cctgcttaca 11760

tccaaactga caccactctt cctccgccca ctcacaacat tcgtttccta cgatgttgtc 11820

attctctcac cctctgctta cgctcaatgg ctcatggcaa aatctgaagg taaagaatac 11880

ggtctctttt tcgctgtcat tcaatcagtc tttgatttta catctcaaca cgaagttgaa 11940

ttgacacttg aagcacaaga gattacacag caagtccgtg ttgaaattca taacaccgaa 12000

cctaaagata ccaaggatga cacctgctac atcacaccca ctactattat taacccaatt 12060

ttaattagaa ctgacaatta cttctacacc aataccaaga aagtgatctg cgaagctccc 12120

attgaattat cagacaaggc taaagaaatc gtcgctaaac aacttgacgc tatcccacat 12180

cctgatttag agacaattga cttagaagtt gccacactta ttgactgcac aactacaact 12240

aagcaatcag aagttaaaga aatcgaagta gatgacttaa cacacgccta tctactcgct 12300

ctcatcaaag caacttgtat agaatctggc ctccccatcc cagtgatgaa ctacatttca 12360

tcccaaaccc aaattgattc agtgatgatc cattacttcg tcaaagccgc taataaacta 12420

ttgcttaaag atcaggaact gcctctcatt acagcccaat ctgacacctc caccattcaa 12480

gaaaccttgg aagctctcaa attaaaggac accaccctaa tgaaccccca agaacttaaa 12540

gctcaccgca aacagattaa cattctgaaa gctgaacaag aacgacagct atccatggaa 12600

cggaaactca acgctttcct agacacaatg caccaacgtg ctgtggctga aaagaataga 12660

tcagaagttc acagcaaagt atcagctgct ctacgcatgc acctttcaaa ggttgcaaac 12720

gctgcacatt gtgacatgga aacaacagac ggtgttatca cactatgctc tgctttctct 12780

attcgctcag tctgcattac tcaaaacact gcagatgtct ctatctacta ctcacaaccc 12840

acagatacat actcaatcac aactaccacc acagtcttca cagcttacaa ccctatgaac 12900

gccaacggac aacccatcat tccaaccact gacccttaca atcctatcat ttcattcccg 12960

gttatcttct cactaaaaca ccgtgaggaa aaatcggaga tagaacaaca agcaaacgtg 13020

ggatactccg aacgtgccat aaaaataaca acaactgaac acgccactgg tacagttctc 13080

atgattgaca acattacatc tgtcatcccc gccactaaaa ccgatgctcg cttcaccgta 13140

aacggtaaag gctacaagat cgtcgacaat gccgttgccc acaaagctcg tgctaacatg 13200

tctacaattc ttgctgtctt gcgcaaatcc accatcagcg cacaagcagc agtagttaga 13260

attggcggtg ctagatacca aacatctcac caagctatct ctttcacccc cttccaaaca 13320

ccaggctatg ttacctacgc aggaatatgc gtctgccaac gctgcacact cagagaacaa 13380

cacgagtgcc actacgcagg taactttgtc cagataccca ctaacatctc gacacaagga 13440

cttaataaaa tttatgaatt aacatctatc ggcccatgca aacacaacca atttgaatgt 13500

accacatgtc aaccagatct aattacaaaa gcccccggac aagtactggt ttacttcaca 13560

gcccacgccc aaacccacta taaacaaata gttactcaaa tctgcaagtt ttaccaagtt 13620

aaacaatgca actcccaaaa gcatgcagat ttcactatcc acataattcc acacggccac 13680

tcaactgata caactcatcc taacaccacc gttctagttg tacgaaaaca cctagtcgat 13740

ccagatcctg aacctccaca catcgggata agttccacct taactggcga agaactgatt 13800

actcttgaca gattactaca acctgctgtc gagcctcaaa ctaagtcgtt tttaaacttc 13860

tggagcgggt ggtaggtgta tcgttaccat ccgctagcga cccttataca tcagggagct 13920

ataaaccgat acctcgtatc ttttacatta acgttgcaac acgtaatgac acaaaattag 13980

ataaagtctc caaactcaac acctggagca tggaaacacc aagcggaatt atcatggtaa 14040

aaaatcgtga cgcctctaaa gaatttgata tagctaacaa cctaccaaag tcactaccaa 14100

ttttaacaca taaactcatg acttacaacc gcgacaacct cctaatcaga tactacacaa 14160

caccttactc actaggagac cttgtctacg cttaccaact cggcgatttt gaacaccttc 14220

tactcgcggc cggactttca ccaaatcctg cccaattgct cgaccctggt aattacaccc 14280

aatatcaaca aacaaaagaa catctcagac aaattatcta cagatctaat ggaaaacctc 14340

cataaaatca tttattactt caccaccact gataaatatc tgccgctaac cctggacaac 14400

attgatcaga acggttattt gtatgatttc ggtgactacc cagctaaaag tcccgtcaat 14460

aatcaccacg tactagaatc atggacacaa ttctgtgtat tctgcgaagt taaccttgct 14520

gaactaccag gttttaacct ctacactcaa acccactcaa ctccacaaat tataaagagc 14580

cgacaagacc tagtctctgg ctcctacatg gataaactac tagctcttaa ccacctaata 14640

gcagcaaatc cagaagcccc ttccaccgct tccactccct tctttaacgc ctccgcagcc 14700

acaactgaca tggtcggcct acactcacca ctctttggct accatgaagt caacaatgat 14760

ttgattcgct ctaaactctc accaacagaa gttgacttca tcagatctca agattctgca 14820

aactatgcta aacccaacgt catctccatc ggcaacacaa ttagctacta cggcaaaaga 14880

ataacaacta aagctgccca aagaaaacct caatacgatc tcgacatgct caaaagatta 14940

gaatcaattg gcatcagcac actcgctaca acatcttacc actaccaagt agctgacgat 15000

gaacaatgtg tgcaagattt taaatactac gattacaact cgcccaaaac ctatgaccct 15060

aacttcctca aggccatcta ccactacatg caagacatct tttccattta caaaactgat 15120

gtttccctta accatgaaac aggacaacca cgcccctctt ctatgggtct tggaatcaca 15180

ggttttactc aaaaagctgt ttggcagtct ctaccacctg attttggccc acgcatgctt 15240

gaagttgcaa aagaaactct actaccattt acaacaaagg taattgcaaa atacgctcgc 15300

acaaagaaag cccgtgcacg tacagtaggt gcttccagct tcgtcgcatc aacagttttc 15360

agatttcttc acaaacctgt aaccatgcgc atggtcgaac aagcccaaaa caacattggc 15420

ccactcctaa ttggcgtctc taaatttaac ggcggttttg ataaatacat ccgtactcaa 15480

catccaaacg gtatagaaca ttacaacgtc atgggtgcag attacacaaa atgtgatcgt 15540

agcttcccat tgatcaaccg cgccttcgcc gcagcccttt tctatgattt aggaggttta 15600

ccatctgaca acatgtggtt tatcaacgaa atatccgcct tcatgactga cttctgcaac 15660

atctcaggtc acacatttaa taaaccaggc ggcacaacat caggcgactc aacaactgcc 15720

ttttccaact ccatctacaa ctacttcgta cacctcactg tacaattcct caccttcctc 15780

tcagccgacg tgccagaaac atacaaacat cttcaaggtt ctgctcatcg cgcttactca 15840

actggaactc tcgaagatta caacacctac ttcgaactca tccatgattt aaactgcact 15900

gaatacttct ttcactttct cagtgacgat tcttttatct tctcaaccca agaagctttc 15960

cccatcttca caacatctaa cttttcaacc aaacttcaat ctatccttgg ctgcactgtt 16020

gacgtaacta aagcctgggc tgcatctgga cacatcgaag aattctgcag ttctcacatt 16080

gaagaaatcg acggtgtcta tcaatacatt cctgatccta acaacatgat cgcaggcttg 16140

tacatgtccg gtgacccagt accagtagat aagaccatct ggaaactagt tgctacactc 16200

agtgaattgg ccatttttca acacaaggac ccatcacttt ttaacgccct ttaccaactc 16260

cttcaaaaca tgcacagtga atatgtcact caacacaacc acgctctcct ccctcttcaa 16320

ttgcttaata agaaattcta cactgatcta cttcaaccag gatttgatac tgaaacaatt 16380

ttcctcactg acttgctaac atctgtcggt gtgaaactcc aagcatctaa tgattcacaa 16440

tgctacttct gtgacaaccc tacaatttca acatgtgctg attgcgtagt gccatatcct 16500

atgtgtgcac tctgcgccta cactcactgg gttcaaacag accatgcccc ctcacaagta 16560

ccaccatgtg tgtcctgtgg ctgtgtcaat cctggaaagc tttaccactg tttcactggc 16620

agcgttaaaa tctgctgtca agatcatgtc tctggcctct ccctacctct agtagaccac 16680

gccaagaaag taattagaat acccctcttt caaaactgcg aacgtcaacg atccctagtc 16740

catgcaataa cacacacggc cttatttgat gacaatgatg aaccacttaa caccgcaaca 16800

ttttccaaat atgatccaga acaaactaaa gaagaaaact tcgtacgcat cttaaacgat 16860

tgctatatgc ttgaagccta ccaagctcac caaaacccag catataccta tgacgttact 16920

gaacctggca caatagtcgt atcacaacct ccatcaacag catatggtaa ctgtgtctac 16980

gctgaaattc tcgatgacaa aaatcgtgtc gtcttgaaag tcactcttga tacaaagtca 17040

gtactaacac ctactgtcta ccatgtaaca tcttgtcgtg gtacacttta cactcgctac 17100

aataaaatcc gcagagttca gcacaaacct tcaatagttc ctagatcaac cctagcctac 17160

tttaacaacg cacaattcat aattggacca ccaggaacag gaaaaactac tttcgtcatc 17220

aagaactaca ttgacagagc agaaccaact aacaaaatag cctacattgc cccaacacac 17280

gcactcgtac aagctatgga tcaagccatc ttcgaagctc acggatatgc agctagtgtc 17340

aacatcgtta aatcagaact taacaacaag acctacaact acaaagcaaa catgcctgga 17400

tgcgatatca tgctcgggac acccggagct gtatcagcgc acactggttg taccatgatc 17460

ttcgatgaga tgtcacttgt acagttgaat actgctatga ctgcaattgc tcgagtaaaa 17520

ccatctaaac tcatccttct cggtgaccca tttcaattag gcccagtagt tcacttcaga 17580

aactttcagt acaactacga agacttcccc tatcttcatt tcgccccaac aataacatct 17640

ctccgcgtat gcttccgatg cccaacccaa atctttaacg tatggagtca ctactacacc 17700

aaacatgaca tcccactcaa tgcctttaaa gacggcggct catttgaaat cattaaacac 17760

cactcatgcg ccagagtttc agaaacagct caccctctgg tcacaagact ctcagaagaa 17820

aatccagact ccatgatcct ttgtaactac aaagctccag tctcctgcct cgaaaacgct 17880

ctaacaattg actcaagtca aggcagaacc taccgagacg tcatagtcgt gttgctcgga 17940

aacacaaatt tcacccgtgt acccaaccga tttattgtcg ctgcttcaag agctacacat 18000

tctctcaaag tttacctttc tgaagagatg tacgaccact actccacact attcaattgg 18060

ccacacttag aaacaccaca aatccagcca caaaacgcct tcacaacacc taacaactta 18120

gaagaaacaa atctcacaat ggccgctaat aacatcagct ctctcgtcat ctgtgatata 18180

gaattcttcc atcaagttcg cagccttgaa cccaaagttt gcacacttga agtcggtgac 18240

attaccatcc atacatcaac aagcgccaaa cagatcatcg ttccacgcgt ttcatcaaca 18300

tctagtgaaa tttttccaaa atactttgct cataacatgc caaccctcac tctccccaaa 18360

ccaacaccaa aaggagattg ggactacatg aaacctcacc gtggaatttc tcgccgcatc 18420

aacatcgaca gaactcacaa actcttctcc catatggctt gcacaacaac tggaactccc 18480

atctatgttc tctacggtgc ccgcaacgat atcctagccc tttctaaatt aaatatcgtt 18540

ggtgactata aatgtttctg caatctacca gctaactact tcaccctaga tgcccaagat 18600

aacatcaaat actactgcac ccaacacgcc aaaccagcag cacctctcgc tggaaccatc 18660

aatgctaaat acattgacat ccaaacagaa aagatcactc ttgccaaacg ccatgctgaa 18720

atttgtgatc accaccatgg tgcagcacac acatctgatg tcgacaccat tatgaccgga 18780

tgcatcttag gcaatgaatt gacactagca actcaacatc tcagccagct tctcttcacg 18840

gaacccttca gccactactc aagtgtaatt tctccagata tgcaccttac agtttcaacc 18900

cgcccttgca ggacactcgg acaacttatc ataactaaca tcaatggtaa gaactacaga 18960

ttctcaatcc aaccacacct ggatcttaaa acacactttt ttaacatcgt tgctaaacaa 19020

ggccactcct ctgttttagt caacgttcca gaaacagttc cgacctgtac taaaattcgc 19080

ggtgaaacct gcgctacatg ctacaacacc tggcttatcc tccaagaact tcatgctgaa 19140

ctagctaaca caggtctcgc cctggtcaac ccactccaac gccaatacac tcaacgagaa 19200

acacaaattt taaccaaggt aatgtctgtc actccaacat ctacaggcaa aatcatggtc 19260

catctcgaca acacactctc cttccccttc ctactcgatt taccaacaag tatccaaaca 19320

tactgtaaca agatcgataa accaatccct aaccctgcaa ttttcacttc ccttaaaatt 19380

acacacactt acggattcgg aacaccattt gtccctatga caaacaaccc actcattcca 19440

tctatcctca gtaaccaaaa acttgctgac aacccagact actactacat cacacatcaa 19500

ccatcaaaca gtgaacttcc tttcatgact tcttcaccaa ctgctggctt ctattgctat 19560

gacacccctt tgattaactt ctcggaacgt accattaaca aaccacccta ctatatcaca 19620

cgaattgtcc gtggccaact acaaaaacct gatattggcc tcttctccac taatagactt 19680

ttcaccaacc aatacgtacc tttacctaat gaacaacaag cgtacgaaga ctttggtaaa 19740

catgtctcac ttggcgaaac aacatcaaca tcctggacaa tcggtggcat gcatctactc 19800

ccagccttcc aaaccacaac caattggcag atcacatcag aacatacacc aattctctca 19860

attaccatag ctcgcgatcg cggggtcaaa ctaggaacaa cagccctaga tgtacctgct 19920

gaccaactac ttgaattaat cgactataaa aacatcacag tctcaaaagt aatagctatt 19980

aagatcgacg gccaaaaagt aaacatgatg gttttcactc aaaacggtaa aatccaaaca 20040

gcctacccag tacaacaagc aaagatgcac gatgtgacaa aacaccacgt cactcctgac 20100

tacataatct ggcctaaaat tttcagcgac tcacctgtcg ataaatggga tcttcctaat 20160

tacaaccaac cacctaaagg tctcacatcc aacgtcaaca tccataaata cgatcaaatg 20220

tgtgcattct ttgacaaaca cctaaccatt ccaacaaatg gccacgtaca ccatcttggt 20280

catgccggac aagtttacag ccctggtgat atagttttaa aaagctattt cacaaaagct 20340

cgtttaacat catacgactt gcgtgattgc atatcaacca gcgaaacagc tcaccctgat 20400

gacaactggc gtgcacactt catcctgtca gatgcatact gtcccaacac cgactttaca 20460

tctctctgca aagactacat gactaaacac ctacgtcttg gcggaagcat gctctggaag 20520

atgacagaaa cctgtaacat cgacgctgaa tccattctac catactttgg atctttcact 20580

ttcgtcacct tcgctgtcaa ctactcatca tctgaaactt acgctttcct taagggatac 20640

atgggtgaac ctaactacaa caccacactt ccagctaaca tcatgggtca actcggcgcg 20700

tacagacaaa acctattgta cgtcccctat gctagtgagt ataccggtac cagagtttat 20760

aaagattctg gtgctgttaa agtcattccg gccatgtatg cagatcgtct gaatacttcc 20820

cactatgctc tcggctgcat ctacagcact tagacatggc aaacttgcta tggctaactc 20880

ttctcatcac tctcagtctc gtgtccgctc aaattaacac gacacttcca gactgtccta 20940

ccccaacctg cacctacaca ccaccaacaa cgaccccatt tgaccacaca tcactacact 21000

cttttttcac acatgacaca aactatgctt tccttcttgc tcttaagtat aatggtactt 21060

tcattcctgc cactatggct atgcctgcta acaacctttc gcaaacatat acactcaact 21120

ataaaaatgt ctttgtcaac gtacctgatt ctcaactccc tcctcctcaa agaagtaata 21180

ctcagttggc tgttgttgca accacagatt cctgtcttac tgcaacgtat caagcgattc 21240

ctggtactat tcctggcctt tcaactaatc acatgtattg tgtgaaattt actcagtcgt 21300

ccaactttgt gtacttgtac gataccactg tgaaaacccc tgttggcgta cttttaaaaa 21360

acccaactgc cccagtagtt acaactatcc tatccactat catggttaac cgccctacaa 21420

cagtttcaac ctcacagctg ctagctaata tctactccct tatgagaacg agaagcaacg 21480

tgattcaaca acaattcact tgcacatctt gcctctttgc catcgacgca tctacaggaa 21540

gagcagctac ctcaccaact acttacctcc taaacaactg gtccccatcc ttctttgaaa 21600

ctaaaacagc tatgttggac cacgtctcta tgaacaccat ctaccaaaca gcacgtggtg 21660

ttactcgtga aaccttcact ggttacatcc ttaacgcagc taaaaccgct gcctacgctc 21720

gtggttctcc cgcctattac atcttttcat tcgcagccat tgcacgctac catcaagcaa 21780

tggttactcc ttataactat aacatcggaa aactttgtga agcaacagct gcttgctgct 21840

tatcaaatgg cattgaacat tttgacatct atacctggct atttcactac tacggcttta 21900

tgaaatccaa agtctacagt gaaactttta caacaaaccc agtactaaca tcagttatta 21960

acttagccac atcaattaaa ggtaaacatt ccgtgtactg tgacctaaca ccttacagtt 22020

catttactgc ctataatttg atcatgcttc aagacgttgt cgaacaatac caagggacac 22080

gtacaacatc cacatacgct aacaattttc tcaacctcac tgatattaac ggatacctac 22140

cctattccac cgataccccc ctcccacaaa actaccaaaa ctacatgcct tttgcaacct 22200

acctacttgc aacattttac tcttctttca ctttacaaca gaaatttgac ttccacagat 22260

tcctccaatc tgtctgttat tatgactctg gtttcatttc tcgtaaccaa gcctaccatt 22320

ctatctgtca cataatcact ctgaaaacat catcttcagt accttacccc gcagaactcc 22380

aatggccctt tactgctctc ccttcctctg gttatgactc ttatccagtt tctacagatt 22440

taggcacagt agctgttggc aacacaaatc cacttgatta ccctggaatt tgttaccaat 22500

cagtttgctt gactcatcct actattaact tcagactttt gttatcagaa ttaccaatta 22560

aatgggaagt ccgacctgaa ggtatctatg taacatctag aaacgatcct acatgcctga 22620

atttcaaaac atcacagttg ggaacagttt tcattacact ctacgattct accactacat 22680

gcacaattca acagactact tactctcgct ctgctgtcct gcgccaagtc aacacgttta 22740

caccaaatgc tactgattta gtcgtgccct acggcaaaac accaatatac aatggagcct 22800

ataatattaa ttccactgca tctggtgctg tctctctctc taccaacaca cactacattc 22860

cacgtctcct cacttcagtt ttaacaccgc aaagtgacta caagattaca cctacaacca 22920

ctaatcttga accattcgtg gaaattaatg gcacaacctt ttactacgat gctaatcaag 22980

cctacgttaa tacatcggac gtttatcaag ctaacgttga ttttggactc ccccaaacat 23040

tttctcttga taccagcttc cttaattgca cagctttcct ctgcgcttct gactacaaat 23100

gtatcgccga cttttcacca atctgctcag ctacacaacc actaatcgac gccctactcc 23160

aaacctacac actgtacaat caacaactta cactgtacaa ttcttccctg ctcacaattc 23220

aagccgctgc aaactacctc tacgacacaa gaacacgtac tagacgatca ctcctatcta 23280

ctgtcaacct tggctacggt gacgtttttc aacaaccagc ctgggtctac gaaataggtg 23340

caacagctgc aattcctatg atcggccctg cagtctcagt cggtttgtta gccagtagag 23400

tagcatctat tgaatcaacc ctctatcaaa ttacccaagg agtgaaggag tctattatgg 23460

ctatgaacac caagcttgat aagattgcca acattttaca ctcagacatc gaaagattag 23520

gattaacagt taacacatta gcctcttcat tcaacacatt cactaaacaa gtaacagata 23580

aatttgcctc tgttgatgtt gcatttaacg acttatcaac atcactgaac acactcacca 23640

caacaacaac tgttggtgcc tcttatgcag ctcaaattaa cttcgcaaca accacgctaa 23700

ccaatttaga agttcaaatc tctcaaacaa cagatcacgc tctatcttgc atcgctgcgc 23760

ttaatgaaca tgttctttct ccacactgca tttccgtttc acaacttagc gcttacacac 23820

ctaatgctct ctctaacatt ggtgaaaaac tagtactaac tcactacatc aacggatcac 23880

tcatcacatt ctaccatctc attgctagca agggagttta cagacccctg ccaaaaatgt 23940

ataattctac acaccattta gttgcaacaa ctggcttcta tgacggccat tgtatcttct 24000

gtggtgaaca ttattgtgat atcagcgttt ctgtaccatg tgattatgcg tacgaaccac 24060

ttaaatccac tgtactagca gcttacccac taacaactgg tgtagccctt ctcactcttg 24120

actctacagc tactaacttc gttattaact ataataatgc ccctctcatt tcatcaagcg 24180

ttccaaaacc acctatcatc aataacaccg atggtacagt tattacgctc caacaacaag 24240

taatccagct ccaaactcaa ttcgatcact ttaacatctc gtcgaatttt cacgtgtcag 24300

aaatccaacc agtcatagac tactacaatg cacaaatcaa ctcatctcta aaccagatag 24360

ttgatttcgg aaatggctca acatctaatg taggaacaat catcctcatt gtaactatag 24420

ttgcccttgc tcttgcatca gctatcgcca tcatctgtgg attaaagcac taaagaccaa 24480

caagttgcaa tggcttccac cggtaaaaac accaccgcag gagatcccaa ccttgggttt 24540

cacttacaac ttaatgatgt ccttaacacc ctggacatct ccggcttcag catctcaaat 24600

tctcatttga tgagtgcgat gctgaccctt aaccttctct ggagcgctat ctttctagtc 24660

tccttctatc taatcttgga catggttatg tactacgttc ccctgctcaa taagattcca 24720

ctgctgccaa caatcaaaaa cctttgttgg tttatctcga ctctgcttgt tgctgtttcc 24780

atttacgctt caggaactct gctgatcatc aaaatcttcg gaggcgtagt cttgctcctt 24840

gcttttctcg gcatggtgaa gactttctat atgctcattg caatggcgct gtcctactac 24900

cgacaccgta acatcgcagt ggcattgaac ggccctaatg tcgttatggt taacggcaca 24960

gcagtccact ctgatttcat ccccacgtct gtgacactgg tgaagaaaga tggtgaactc 25020

tattttcaag gaacaaaggt ccccggaaac aacttgattg atgcaaccgt gaagatcttc 25080

actttcaacg aatcgtttga atgcctcccc atcgcactct cacgttacac taaaggttcc 25140

tacgctagat ttgagctcaa gtcgaccaac acccactatg aactccaaac tttctatcct 25200

tcgcaaacag aaactaaatc tgtttgaaac actagcgaaa acaaattaca atggcattct 25260

acggacaacc aatgatgcct atgcctatgc ctgcttcctt tcctcctcgt cgccgtgccc 25320

ctaaaaagcg cccgccacag aagaaacaag ccaagcccca acctaaacct ctcaatgacc 25380

aacagaaaac agctgttgcg aaagaggcta ttgccaaaac caatactgtg ccaatgccac 25440

ccatggccac agtagtgaag acaaacctgc gcgttcaggt tgtaaaaagc aaacgcggtg 25500

acctcctaga tgctcgaggg cgtcttacac aatcagagat caaccagctg tatttccaaa 25560

tcgccactgc cctcaacaac ggcctcggag aaattggaat acataatgct tcaacgcagg 25620

acggcgttcg tctcatcgtg cgcctcaatt tagcccctcc taatcctatt gctgagaaac 25680

tctacaaatc tcacgcggcg gctaatctca actcagaaat tctggtttct aaacaaaagg 25740

aactggagcg agaccccatt tttcatgttt cctcgactga gtaaactgac ttcacctcaa 25800

aactgaagaa agtatatata atcacatctc tcacactatc ctttataaac tatatattgg 25860

ggtggcatta ggccattcca aagaattctc taagatcaca ctcctcgata cctgactagt 25920

catcacgttt cctggggtct gctctttaac attctactat cctataagat ctaaaactat 25980

ccaaaaaaaa aaaaaaaaaa 26000

<210> 2

<211> 389

<212> DNA

<213>Artificial sequence

<400> 2

tgaaacaatc caagaagaag aagatatcga agaggaagta acatctcctg ttcaacccac 60

actcaacaca ctccaagaac aagctactct caacgaatcc attcaatcac tcgttgaatc 120

agttgattct gatgtccctt acacctggga acaacgtgtt gaagaaagtg aggttgcaac 180

tattgattta caagctgtag aattagatca aacacccact cccaccactc tctcaacaga 240

agcagtgcaa gaatgggtca acgcaacatc acctgacctt gctacagtag actggaacag 300

tgcaataact gaacaggaaa ccacagtaac ttcactaata gaagaaatct ccctacgccc 360

caacaccaac aacaccggct ataacttgt 389

<210> 3

<211> 19

<212> DNA

<213>Artificial sequence

<400> 3

atcaaacacc cactcccac 19

<210> 4

<211> 18

<212> DNA

<213>Artificial sequence

<400> 4

aggtcaggtg atgttgcg 18

<210> 5

<211> 25

<212> DNA

<213>Artificial sequence

<400> 5

tgacccattc ttgcactgct tctgt 25

Claims (3)

1. a kind of crucian coronavirus HB93 TaqMan real-time fluorescence quantitative RT-PCR detection primers, which is characterized in that described to draw The sequence of object is：Forward primer F：5'-ATCAAACACCCACTCCCAC-3', reverse primer R：5'- AGGTCAGGTGATGTTGCG-3'；The deposit number of the crucian coronavirus HB93 is CCTCC NO：V201722.

2. application of the primer described in claim 1 in crucian coronavirus HB93 detection kits are prepared, crucian hat The deposit number of shape virus HB93 is：CCTCC NO：V201722.

3. application according to claim 2, the kit includes positive control, and the positive control is by SEQ ID It is obtained in the insertion pGEM-T carriers of sequence shown in NO.2.