CN109628638A - Detection method and its application based on prawn east virus genome sequence - Google Patents
Detection method and its application based on prawn east virus genome sequence Download PDFInfo
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Abstract
The present invention provides a kind of detection methods and its application based on prawn east virus genome sequence.The prawn east is viral (Orientavirus penaei), belongs to bunyavirus, and deposit number is CCTCC No.V201864.Form is spherical in shape, diameter 80-120 nm.Can infection of Chinese prawn, cardinal symptom shows as vigor reduction, and hepatopancrease shoals.The present invention also provides the detection kits of above-mentioned prawn east virus.New possibility is provided for the detection and prevention and control of shell-fish aquatic products bunyavirus.
Description
Technical field
The invention belongs to aquaculture fields, and in particular to the detection method of prawn east virus genome sequence and its answer
With.
Background technique
The bunyavirus mesh newly named in the tenth report of International Commission on Virus Classification is divided into 9 sections 13 category, section's difference
Viraceae (Feraviridae), ficus virus 1 section (Fimoviridae), Hantaan virus section are drawn to take
(Hantaviridae), the more Viraceaes of rice card (Jonviridae), Nairovirus section (Nairoviridae), general bunyavirus
Section (Peribunyaviridae), phantom Viraceae (Phasmaviridae), white fine Viraceae (Phenuiviridae) and kind
Eggplant spotted wilt virus section (Tospoviridae).
Expense draws Viraceae to only have 1 Tobamovirus: positive expense draws Tobamovirus (Orthoferavirus), main infection mosquito, no
Vertebrate cells can be infected, the restricted virus of insect host is belonged to；Ficus virus 1 section includes Yi Mala Tobamovirus
(Emaravirus), can infection plant, and the arthropod-bornes such as plant graft or mite class can be passed through；Hantaan virus section includes just
Hantavirus (Orthohantavirus) can be propagated through rodent and insectivora animal, also infect the mankind once in a while；
It includes the positive more Tobamovirus of rice card (Orthojonvirus) that rice, which blocks more Viraceaes (Jonviridae), can be in a variety of mosquito cells
Duplication amplification, but vertebrate cells cannot be infected, belong to the restricted virus of insect host；Nairovirus section includes just interior sieve disease
Poison belongs to (Orthonairovirus), and the coe virus is most of to be propagated using tick, mosquito as medium, and fractionated viral can also be in louse, midge
It propagates in equal arthropods, or is propagated between different hosts by tick worm.Nairovirus section is in Africa, Asia, Australia
Continent, Europe and America are distributed, and infectiousness is strong, lethality is high, can cause a variety of serious mankind and animal infectious disease
Disease；General bunyaviridae is made of conspicuous primary Tobamovirus and positive Bunyavirus (Orthobunyavirus), wherein positive cloth
Buddhist nun's subviral can infect people and ruminant, can cause such as fever, encephalitis and Hemorrhagic fever serious disease of patient；Phantom virus
Section includes positive phantom Tobamovirus (Orthophasmavirus), only infects cockroach, geometrid moth and mosquito, belongs to insect host limitation venereal disease
Poison；White fibre Viraceae is by lattice gram Tobamovirus, the western Tobamovirus of pa (Phasivirus), Phlebovirus (Phlebovirus) and fibre
Thin Tobamovirus (Tenuivirus) composition, lattice gram Tobamovirus are only capable of infected mosquito, and there has been no other vertebrates of infection and segmental appendage are dynamic
The report of object；The western Tobamovirus of pa can infect the arthropods such as fly and mosquito, and there has been no the reports of infection vertebrate；Sand fly virus
Belonging to virus is all arboviruse, can infect a variety of mammals and people by arthropod-bornes such as sand fly, tick, midge and mosquitoes
Class seriously affects human health；Very thin Tobamovirus is a kind of plant virus, can be expanded in plant cell, and plant hopper, leafhopper are passed through
It propagates；Tomato spotted wilf virus section includes positive Tospovirus (Orthotospovirus), can be passed by plant leaf
It broadcasts, can be propagated between host plant by insect vector thrips.
1996, Australian scholar in the morbidity Penaeus monodon in northern Queensland state gross profit town (Mourilyan) for the first time
It is viral (Mourilyan virus, MoV) to identify one plant of bunyavirus-gross profit, the virus is then in the big benefit in Southeast Asia and Australia
It is had been reported that in sub- japonicus and Penaeus monodon, it is pathogenic that existing research proves that gross profit virus has japonicus.2018
Australia scholar Sakuna was reported and was had found one plant of new bunyavirus, phylogenetic evolution tree from red claw crayfish year
Show the positive Bunyavirus (Orthobunyavirus) of the virus Yu general bunyaviridae (Peribunyaviridae)
Affiliation it is nearest, and speculate that the high mortality after the long-distance transport of the virus and red claw crayfish is related.Currently, discovery can be felt
The bunyavirus for contaminating Crustaceans is limited.
Summary of the invention
Aiming at the problem that current shortage detection and prevention and control shell-fish bunyavirus product, the present invention provides one plant of prawn east
Side's virus (Orientavirus penaei), belongs to bunyavirus purpose novel species, can infect shellfish.
Another object of the present invention is to provide a kind of kits for detecting above-mentioned novel bunyavirus strain.
To achieve the above object, the present invention adopts the following technical scheme that.
One plant of prawn east is viral (Orientavirus penaei), belongs to bunyavirus mesh, December 06 in 2018
Day is preserved in China typical culture collection center (CCTCC), and preservation address is Wuhan University's collection, and deposit number is
CCTCC No.V201864。
Prawn east virus is spherical in shape, is made of the minus-stranded rna virus of cyst membrane, capsid and three segments, and diameter is
70-120nm.Can infection of Chinese prawn (Fenneropenaeus chinensis) etc., cardinal symptom shows as vigor reduction,
Hepatopancrease shoals.
The genome sequence of prawn east virus is as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3；Its
In, the sequence of the segment L is as shown in SEQ ID NO:1, and the sequence of the segment M is as shown in SEQ ID NO:2, the sequence of the segment S such as SEQ
Shown in ID NO:3.
A kind of kit detecting class prawn east virus, class prawn east virus refers to same with prawn east virus
The virus of category.
The kit is the examination for carrying out detection kit using nucleic acid amplification method or being detected using immune response
Agent box.
Further, the kit is to carry out detection kit using nucleic acid amplification technologies.Since east virus is one
Kind single strand RNA virus, the synthesis of geneome RNA is directly replicated by its RNA polymerase dependent on RNA, in rna replicon
In the process, the error correction that 3 ' -5 ' are lacked dependent on the RNA polymerase of RNA, causes genome to be easy to happen when replicating naturally
Mutation, the segment of the specific sequence in whole or in part of genome is interior, and there may be >=70% variations, therefore when detecting, also
There need to be detectability for >=70% homologous sequence.
Mentioned reagent box with
(a) RNA sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or corresponding DNA sequence dna all or
Partial Fragment；Or
(b) complementary series of (a)；Or
(c) (a) or 70% or more homologous sequence (b) are specific sequence.
Above-mentioned specific sequence length is preferably 12-3000nt；It is furthermore preferred that length is 30nt-1200nt；Most preferably
, length 40nt-600nt.
The kit includes 1 pair or 1 pair or more of pairs of primer.
The primer of the kit can be the specific sequence of detection sequence or the sequence of the base containing degeneracy, can also be
Sequence comprising lock nucleic acid.When detecting for right >=70% homologous specific sequence, simplest mode is to will be present
The multifarious mutational site of mononucleotide is considered as the degeneracy site of corresponding base, thus the available letter comprising various mutations base
And the sequence of base is as primer sequence；This degeneracy mode can also be replaced with hypoxanthine or other equivalent bases.
The length of the primer is 9nt-45nt；Preferably, length 15nt-30nt；It is furthermore preferred that length is 18nt-
25nt。
Further, the primer is selected from any 2 or 2 or more as shown in SEQ ID NO:4-SEQ ID NO:26
Forward and reverse complementary series sequence or 2 between SEQ ID NO:1 specific sequence or its reverse complemental spy
Anisotropic sequence；Or as shown in SEQ ID NO:27-SEQ ID NO:34 any 2 or 2 or more forward and reverse it is complementary
The specific sequence of SEQ ID NO:2 between sequence or 2 or the specific sequence of its reverse complemental；Or such as SEQ ID
Between any 2 or 2 or more forward and reverse complementary series sequences or 2 shown in NO:35-SEQ ID NO:40
SEQ ID NO:3 specific sequence or its reverse complemental specific sequence.
When comprising 1 pair of primer, the real-time fluorescence quantitative PCR of Standard PCR detection, SYBR Green I dyeing can be used
Detection, digital pcr detection or the isothermal duplication (HDA) dependent on unwindase detect.
When comprising 1 pair or more primer, sleeve type PCR can be used or multiplex PCR detection, sleeve type PCR nested are drawn using 2 Duis
Object, wherein 1 pair of primer in outside is expanded as the outer primer of sleeve type PCR for the first step, 1 pair of primer of inside is as shell type
The inner primer of PCR is expanded for second step；Multiplex PCR uses 2 pairs or more to mutually independent primer, to multiple sites or not
The prawn east virus of homogenic type or its variant are detected.
Can also according to ring mediate isothermal duplication (LAMP) primer design requirement, using above-mentioned 1 pair of primer as F3 with
B3 primer selects 4 sequences on the inside of it, by assembling design at FIP and BIP primer, detects for LAMP；It can also be in list
Chain ring region is designing 1 pair of primer, as LF the and LB primer of enhancing amplification, detects for faster LAMP.
In design of primers, can also the 5 ' of primer terminate the artificial sequence of upper 3nt-50nt, anchor series or other
Implementation sequence carries out other augmentation detection reactions by the detection of anchor primer multiplex amplification or genechip detection principle.
The kit may also include nucleic acid probe.The sequence of the nucleic acid probe is selected from such as SEQ ID NO:4-SEQ ID
The specific sequence of SEQ ID NO:1 between forward and reverse complementary series or 2 shown in NO:26 or it is reversed
Complementary specific sequence；Or the SEQ ID as shown in SEQ ID NO:27-SEQ ID NO:34 between sequence or 2
The specific sequence of the specific sequence of NO:2 or its reverse complemental；Or as shown in SEQ ID NO:35-SEQ ID NO:40
The specific sequence of SEQ ID NO:3 between forward and reverse complementary series or 2 or the specific sequence of its reverse complemental
Column.
Nucleic acid probe can carry out all nucleotide or spy with digoxin (DIG), fluorescein or radioactive isotope etc.
Determine nucleotide, such as DIG-dUTP, incorporation methods label；It with DIG, fluorescein, reporter group, fluorescent quenching group or can also put
Injectivity isotope etc. carries out end modified label, such as 5 ' end flag F AM, HEX, VIC of probe etc., 3 ' end label quenching groups
TAMRA etc.；The genomic nucleic acids for carrying out the homologous virus directly to prawn east virus or other variations carry out independent original position
Hybridization or dot hybridization detection, can also combine with Real-time quantitative PCR, progress TaqMan probe or Beacon probe it is glimmering
Fluorescent Quantitative PCR detection.
The detection method of the kit include but is not limited to Conventional polymerase chain formula reaction (PCR), constant temperature convection current PCR,
Tao Shi PCR, real-time fluorescence PCR, constant temperature convection current real-time fluorescence PCR, digital pcr, dot hybridization, in situ hybridization, ring mediated isothermal
It expands (LAMP), rolling circle amplification (RCA), single primer isothermal duplication, the isothermal amplification technique (HDA) for relying on unwindase, hand over
Pitch primer amplification technology, nucleic acid express delivery isothermal detects amplifying technique；May also comprise but be not limited to it is right to 1 plant or a kind or more simultaneously
Shrimp east virus and multiplex PCR, multiple real time fluorescence PCR, genetic chip, the chip of its homologous kind or variation kind detect；May be used also
With include but is not limited to simultaneously to comprising prawn east virus and its homologous kind or variation kind multiplex PCR, multiple real time fluorescence
PCR, genetic chip, chip detection.
Above-mentioned detection method, according to the needs of its method, can operation to corresponding method and laboratory room managing carry out
Ability certification is carried out to the commercialization detection service for including prawn east virus and its homologous kind or variation kind, or to commercialization
Seedling fostering, quarantine of passing in and out, Quarantine on production site etc. carry out to include prawn east virus and its homologous kind or variation kind detection；
Corresponding reagent and tool can also be assembled, be assembled into kit form carry out to include prawn east virus and its
Homologous kind or kind of the commercialization sale or application service for the kit of detection that make a variation；It can also be on the basis of above-mentioned detection method
Corollary equipment is researched and developed, or the detection range of existing corollary equipment is expanded into covering prawn east virus and its homologous kind or variation
Kind, carry out the commercialization sale or application clothes to the detection device including prawn east virus and its homologous kind or variation kind
Business.
The kit is the kit detected using immune response.
Further, mentioned reagent box with
(a) as the complementary series of nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or its >=
The polypeptide or protein of the complementary series translation of 70% homologous nucleic acid sequence are all or part of antigen；Or (b) (a)
Antibody is detectable substance.
The antigen can be whole Strain, cracking ingredient, capsid, polypeptide, engineered protein or polypeptide.It is described anti-
Body can be polyclonal antibody, hybridoma, monoclonal antibody or single-chain antibody.The antigen or antibody can be according to existing
It is prepared by the method for technology.
The kit, competitive, indirect or interlayer type immune response, which can be used, can also be used solid support or immune
The precipitation method.The antibody or anti-genic fragment of the prawn east virus can be used the prior art and is marked, and such as fluorescent marker is changed
Learn luminescent marking, radioactive label or enzyme label.The method that amplification probe signal can use biotin and Avidin, enzyme label
It is examined with the immunity test of mediation, such as ELISA.
Such as, a kind of kit detecting prawn east viral antigen, contains in kit: the anti-prawn east virus of solid phase is anti-
The coated orifice plate of body, confining liquid, mouse secondary antibody, secondary antibodies enzyme conjugates, TMB developing solution, colour developing terminate liquid, Tissue Lysis
Liquid, concentration washing lotion, sample diluting liquid, positive reference product and negative reference product.Wherein, the coated hole of anti-prawn east antiviral antibody
Plate is coated with using anti-bunyavirus antibody in advance；Confining liquid is human serum albumins；Sample diluting liquid is PBS；Colour developing
Terminate liquid is the concentrated sulfuric acid；Concentration washing lotion is Tween-20；Secondary antibodies enzyme conjugates is horseradish peroxidase-sheep anti mouse IgM enzyme
Conjugate.
Protection scope of the present invention also wraps the antiserum for stating prawn east virus, polyclonal antibody, hybridoma, list
Clonal antibody or single-chain antibody.The antiserum, polyclonal antibody, hybridoma and monoclonal antibody are with prawn east virus
Strain, Strain cracking composition, Strain engineered protein or polypeptide be immunogene according to the prior art side
It is prepared by method.
Such as, the antibody of the anti-prawn east virus is polyclonal antibody, can by the prawn east Strain of inactivation or
Its specific antigen segment, such as: the antigen protein of L, Gn, Gc, N and applicable adjuvant, such as: being bestowed after Freund's complete adjuvant mixing dynamic
Object, e.g., mouse, rabbit, fowl (egg), pig, goat, ox, aquatic livestock are to carry out initial immunity, behind appropriate time interval, optionally
With after inactivating virus liquid and adjuvant appropriate bestow secondary immunity, be even immunized three times or more it is time immune, to improve antibody
Potency.Behind appropriate time interval, the serum of immune animal, i.e. antiserum are acquired, prawn east disease is further obtained after separation
The polyclonal antibody of poison.
For another example, the antibody of the anti-prawn east virus is monoclonal antibody, can by the prawn east Strain of concentration or
Its specific antigen segment, e.g., the antigen protein of L, Gn, Gc, N bestow animal, and e.g., mouse can optionally add adjuvant appropriate,
Such as: Freund's complete adjuvant is to carry out initial immunity.Behind appropriate time interval, optionally with the virus of inactivation (or antigen protein)
And adjuvant appropriate grants secondary immunity.Behind appropriate time interval, acquire the serum of immune animal, to assess be suitble to
Acquire the mouse of spleen cell.From applicable the mouse acquisition spleen cell and myeloma cell, such as: FO cell strain, NS are thin
Born of the same parents' strain carries out cell fusion with PEG.After the hybridoma cell strain of secretion capacity is provided in screening in fused cell, it is thin to obtain fusion
Born of the same parents system, the fused cell system can secrete the monoclonal antibody of anti-prawn east virus.
The invention has the following advantages that
Prawn east virus of the invention is a kind of new bunyavirus, can infection of Chinese prawn；There is provided based on cloth Buddhist nun
The kit of subviral novel species can detect it.For shell-fish aquatic products bunyavirus detection and prevention and control provide it is new can
Energy.
Biological deposits information
Prawn east virus (Orientavirus penaei), deposit number be CCTCC No.V201864, depositary institution: in
State's Type Tissue Collection (CCTCC), preservation address: Wuhan, China Wuhan University collection, preservation date: 2018
On December 06, in.
Detailed description of the invention
Fig. 1 is the electron microscopic picture of prawn east virus；
Fig. 2 is the phylogenetic evolution tree of prawn east virus；
Fig. 3 is Chinese prawn lymphoid organ pathological analysis electron microscopic picture, visible viral inclusion body in lymphoid organ；
Fig. 4 is Chinese prawn gill filament pathological analysis electron microscopic picture, visible viral inclusion body in the gill filament；
Fig. 5 is 1% agarose gel electrophoresis figure for detecting prawn east virus.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
Separation, purifying and the identification of 1 prawn east virus of embodiment
1.1 separation and purifying
(1) 2-3g prawn carapace is taken to be added in centrifuge tube, the TNEP buffer of pre-cooling and 200 μ LPMSF aqueous isopropanols,
10s is homogenized with homogenizer 10,000rpm under ice bath；
(2) homogenate 10,000g at 4 DEG C of supercentrifuge is centrifuged 30min, retains supernatant；
(3) fragment of tissue, bacterium and other impurities successively are filtered out using the filter in 0.45 μm and 0.22 μm aperture；By supernatant in
100,000g is centrifuged 180min at 4 DEG C of ultracentrifuge, is impregnated and is slowly shaken with TN buffer, and precipitating is made to suspend, and draws 10 μ
L is observed for Electronic Speculum；
(4) according to TEM scanning result, be laid with from top to bottom in ultracentrifugation pipe sucrose concentration (W/V) be 20%, 31.5%,
43%, 54.5%, 66% concentration gradient is added supernatant and carries out 4 DEG C of 130,000g centrifugation 170min after 4 DEG C are placed overnight,
The sample for collecting glycerol and 54.5% sucrose density after desugar, obtains virus, and temporary designations are prawn east virus
(Orientavirus penaei)；
(5) whole genome amplification of virus sequence: in the base for obtaining part novel cloth Buddhist nun subviral sequence by high-flux sequence
On plinth, design primer carries out the amplification and verifying of long segment to full-length genome on this basis, then uses SMARTER RACE
CDNA Amplification Kit expand S segment 3 ' ends and 5 ' ends to obtain whole genome sequence, such as SEQ ID
Shown in NO:1-SEQ ID NO:3.
1 prawn east viral genome sequencing primer of table
| Primer | Sequence 5'3' |
| L-1F | ccgggtgtgttctaatgaatctac |
| L-1R | catggatgaaggaaatgggtg |
| L-2F | atattgggacgccccctc |
| L-2R | cgactctgatggagactacctgtt |
| L-3F | ctgtttcctccggggtatctc |
| L-3R | cgatcagtattgtagcagtgcctt |
| L-4F | cctagtgtgttattcacacaagcatt |
| L-4R | gttcaagctgcctgaggaacat |
| L-5F | ctatgaaaccatgcatgtaaccag |
| L-5R | tttgaggtacatgaaatacgtgtgtt |
| L-6F | acataagtaccctacatagtttaggttcac |
| L-6R | tcacagagtggacatagccttagaa |
| L-7F | gacattcagcttattactatcgagga |
| L-7R | cttatgactatgagctgagggagag |
| L-8F | tcatcctcctcatggaaagatactc |
| L-8R | ggtgttgtagtgttgcattggaa |
| L-9F | agatgcccttgttaccccga |
| L-9R | agccatgcttcagtcaattcagt |
| M-1F | gtaatcaatgatcgtttagttggtctt |
| M-1R | atctttacagtgggattggaggtt |
| M-2F | tccaagtccttaacatagaagcagtc |
| M-2R | tatggattaggggcaacattgac |
| M-3F | ccaggagaaacagtgtggattg |
| M-3R | gcttgctgccaacaatgct |
| M-4F | ttggggtgatggaataggttg |
| M-4R | cgggagctacaagtctgccat |
| S-1F | acagtggtaagaaggcagacaac |
| S-1R | gagagcccagggtgataaaca |
| S-2F | gacacacagatacacgcacacatac |
| S-2R | atgccatgggccttggata |
| S-3F | tgaaggaagtggcagcagagt |
| S-3R | taacaatcagaatgcttatggtttg |
1.2 Species estimation
Prawn east virus electron microscopic picture is as shown in Figure 1, the virus is spherical in shape, diameter 80-120nm.According to bunyavirus mesh
The amino acid sequence for the RNA polymerase (RNA-dependent RNA polymerase, RdRp) that each section's (category) viral RNA relies on
Column, are compared using Muscle software, utilize Mega software building maximum similarity chadogram (Fig. 2), system development chadogram
Show that the virus and the affiliation of the white fine Viraceae (Phenuiviridae) of bunyavirus mesh are nearest.Select white fine virus
The RdRp sequence of known strain again pulls up maximum similarity chadogram in section, shows that the virus belongs to the white fibre of bunyavirus mesh
A new kind of the classification position between the western Tobamovirus of pa and Phlebovirus in Viraceae (Phenuiviridae).To its into
Preservation is gone, deposit number is CCTCC No.V201864.
2 prawn east virus of embodiment infects Chinese prawn
Morbidity Chinese prawn sample preparation homogenate is taken, be sterile filtered and after ultracentrifugation, passes through reversed bowel lavage or muscle note
It penetrates and carries out artificial liver support to it, for the death rate in infection prawn 10 days up to 100%, cardinal symptom shows as vigor drop
Low, hepatopancrease shoals.The lymphoid organ electron microscopic picture of the Chinese prawn of prawn east virus is infected as shown in figure 4, gill filament pathology
Electron microscopic picture is as shown in Figure 5.By picture it is found that in the lymphoid organ and the gill filament of Chinese prawn visible prawn east virus disease
Virion.
The PCR kit of the detection prawn east virus of embodiment 3
The PCR kit for detecting prawn east virus includes following component:
| Ingredient | Concentration |
| Ex Taq Buffer (no MgCl2) | 10× |
| MgCl2 | 25mM |
| dNTP | Every kind of 2.5mM |
| L-2613F | 10μM |
| L-3509R | 10μM |
| L-2688F | 10μM |
| L-3030R | 10μM |
| Ex Taq enzyme | 5U/μL |
| Aseptic double-distilled water | - |
The primer sequence and amplified fragments of the detection prawn east virus of table 2:
It is detected referring to following methods:
After the suspected infection tissue homogenate of Chinese prawn is extracted, RNA is extracted, adjusts RNA concentration to 500ng/ μ L, then reverse transcription
For cDNA, sleeve type PCR amplification then is carried out using primer as shown in Table 2.Specific reaction system and condition are as follows:
The large volume pre-composition in addition to Taq archaeal dna polymerase is prepared, packing is stored in -20 DEG C.Before detection, draws and premixed without enzyme
Liquid is proportionally added into respective volume Taq archaeal dna polymerase, and 24 μ L/ pipe is dispensed after mixing.First round amplification program: 94 DEG C of denaturation
2min；94 DEG C of 20s, 53 DEG C of 30s, 72 DEG C of 60s, 30 circulations；72 DEG C of extension 7min.Second wheel amplification program are as follows: 94 DEG C of denaturation
2min；94 DEG C of 20s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C of extension 7min.
3 first round of table PCR amplification system
| Ingredient | Volume (μ L) |
| 10 × Ex Taq Buffer (no MgCl2) | 2.5 |
| MgCl2(25mM) | 2 |
| dNTP(2.5mM each) | 2 |
| L-2613F(10μM) | 1 |
| L-3509R(10μM) | 1 |
| Ex Taq enzyme (5U/ μ L) | 0.1 |
| Aseptic double-distilled water | 15.4 |
| Template: cDNA | 1 |
Table 4 second takes turns PCR amplification system
| Ingredient | Volume (μ L) |
| 10 × Ex Taq Buffer (no MgCl2) | 2.5 |
| MgCl2(25mM) | 2 |
| dNTP(2.5mM each) | 2 |
| L-2688F(10μM) | 1 |
| L-3030R(10μM) | 1 |
| Ex Taq enzyme (5U/ μ L) | 0.1 |
| Aseptic double-distilled water | 15.4 |
| Template: first round PCR product | 1 |
PCR product is subjected to electrophoresis with 1% Ago-Gel, as a result as shown in Figure 5.Swimming lane 3 and 4 can be observed in the upper figure of Fig. 5
To the first amplified band of 897nt.It can be observed that first, the secondary amplified band of 343nt in Fig. 5 following figure.In Fig. 5 following figure,
Swimming lane 3-10 is the amplified band for infecting the sick shrimp of the virus.
Sequence table
<110>Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science
<120>detection method and its application based on prawn east virus genome sequence
<130> 20181115
<160> 40
<170> PatentIn version 3.5
<210> 1
<211> 6317
<212> RNA
<213> Orientavirus penaei
<400> 1
acacaaagac ggguguugua guguugcauu ggaaaaauca agguuuuaau cucucaauua 60
uggaagagug gcuaagugug uuggagucua acaacacagg ugcuuaugua cacaacccuu 120
cugucaucuu ugagcccaca gacagcccug agcuauugac uuacgaaauc aagaacccag 180
acuccauugg ccuugugacu caaguggaga uugaguuuga ugauaggcca gaugagggca 240
cggggaguac auuagccacu caaguccucu cagugcagag gguuaggaca uuucuucaug 300
auuucaccua uucucacaua ucccacagua cagaugugcg cuuggacaca guuuucacuc 360
caauggggaa uagaggugau caucucaccc cugacguuau ucugagagau gguaacaaga 420
uucuuguugu ugaguuugcc acaacacgag guggggacgc agcacuagaa agguccuuca 480
ggacuaaaac agccgcuuau gacuaugagc ugagggagag ggcugagaaa aggcauucga 540
aucacuucga cucucaggug uucuauggca ucauugugac aaaugagcug aaggugugca 600
guaaccugcc ucugacagaa gggcagguga augagcuggu cuacagauau aggauggcaa 660
uagacauuca gacugaguug ucuggagcau ggggaguauc uuuccaugag gaggaugaug 720
aaaugucaaa gacaacugca gaagucaaga ccauucuuaa gaacauuccc uuaagcuucu 780
cauuugaugg uaaguacaua aacaaggaag uauaugacaa cucauucgga gcgccugaca 840
cagauuaccu gcagaaggug auuggacgac ugaugacaga ggguagagcu gaagccauga 900
agagcggggc agagaugauc uucugugaug agagcaaaac augggguggu gagacaguga 960
gccguaauuc cucugagugc uuugaggcaa ucaagcaaua ugaaggagag cugaugaaag 1020
gggauaggca gcacagcagu acaaaggcaa uuguccagcu gccagcuugg acugcaauaa 1080
gagaagaguu cccgggcucc acaucugucc uaaguuuuga cauuggggag cacgcgucag 1140
caacacaugc uguguggaag ucugccaugu ccgcugugga ggcuuggggg ggguucaaag 1200
acgaugacga gucagagaua augaugacug aagaagaaga ucaggaucau cacaagccgc 1260
acagguccaa guaucacaga guggacauag ccuuagaaga ccacguaagg gccagcuuag 1320
cugugaaugg ugucuucggc aaggaauuca gacggaauga guuuguggca gcucggagaa 1380
gugaaaagcg gaagccuuuc ucucuaacug uugacugcuc uuguguggac cuuuuccucg 1440
auaguaauaa gcugaauguc gagcuagaca cuccgauuca ugguaacuua guucaauuga 1500
uuuccucauc uacugagaug cauucccaag augagcauca gaggaagugu aucagcaagu 1560
ucaugaaguc ucaguugggg guguauacau caaucaugac ugauguggcg acagagcugu 1620
gugucagccu gaagcagcac gugaagagaa accaaaugau aauuaagaag cuuagauacu 1680
auccuguuua caugcuaauu uacccuacug acucauccaa gcacauauuc gucucacucc 1740
uggcugaaag agggaaagug ucaguguggg acacgacagg guguuucaaa accagcaugu 1800
cgaaugacaa ggucguguuc acugacuuug uuucuuuuaa caccucgaaa uuggcuaacc 1860
ugugcaagcu agagucaguc auguauaaua caucaauguu cugggcagag gagaacggug 1920
aggcaagcuu cauuggggau gaaaucaugc caugguaugg caagaccucu agaacucagg 1980
aggaggucug gaagaugauc ucucucuguu ugcucguggc caugucugac aaaaggcaug 2040
uugaagagca gcuauccuug uacagguaca ugaacaugga ggccaugacg ucuuucccaa 2100
gggugccgaa gccauacaag cugcugaaga aaaugaccuu gguuccgaga uccaagcuug 2160
agcucuggau aaucaagaaa cauuugaggu acaugaaaua cguguguucc ggccaucaug 2220
ccauuaagau gaagggagac acaggagaca ccacuuggca uaaccuuauc aaucccuaca 2280
cguuguuucc acucaccucc aucuccucaa acgugaaccu aaacuaugua ggguacuuau 2340
guaauaagga ggagucagcu gagggaaaca cagcggccaa caucuaugag aagauucuag 2400
cucuggagga ugaacuacca gaugucaaca cguuccucgg ccuggaagac ccugaugauc 2460
cuggguacca ugaauucucu ccaagucuuc uaaaggcagc aguugacaau auaaaggaca 2520
gauuuagaag gaccuuugga agcucauggg aggccaacau agacaauaag auucugagag 2580
cucuuggcag cauaacuuua gaggaaaugg ccacgcugaa agcuuccucg aacuucucuc 2640
cugaauacua ugcuuugaag ccaggagaug cguauaaaag aaaaaaagcc auccaaggag 2700
ugagagacuu gauggauggg gagucuacaa ugauguauga gguccugagu aagugccucu 2760
caaaagugga gcaagauggc ugcaugcaca uagauauuuu caagaagccu caacacggag 2820
gagauaggga gauuuauguu auggacaugu caucaagggu uguccagcua ggguuagaga 2880
ccauugcuag aaccuacugu ucuuucauag acucagaggc aaugacucac ccgaagagca 2940
aguucaagcu gccugaggaa caugaaagga augcagagaa acgcuugggu acucauguga 3000
cguucuguca gagcgcagau gcuaggaagu ggagucaagg gcaccauguu gcuaaguuca 3060
ugcagaugcu augcaggcua acuccuaguu acauacaugg uuucauagug agagcgugug 3120
cgcucuggac gaaaaggcga auaaugaucu caccagcucu gauaaaggug uuuugcgaaa 3180
gcacuagcuu uacuucaggc aacgauaucg ugcagaggau guaugauggg uauuuaggua 3240
aaggaacaga gaaguggaua acccagggca uguccuacau acagguuggc aguggcauga 3300
ugcaaggcau ccuucacuac acuucguccc ugcugcacuc aaucuuucaa gaguuucuaa 3360
ggagcuugcu gagaucucau uauagggcac gaagccugcu gauaggggaa cccaugauca 3420
cugugggcca gagcucugau gacuccuauc ucaugguuac uuuuagaacg agggauucug 3480
aaacaugggc ugaggcagca cuugagucau caauggucca ccaccucaag uccacauuga 3540
guagauuccu ggggaucuau gacucugaga agacugcuag cauguugaug ucugucaugg 3600
aguuccuuag cgaguacaug gaaggcgcca gccuacauag ggcaacugug aagucagugu 3660
uugcuugcuu aucagugagu gagcaugaga gccugucuuc ucgccaggaa gaaaugagca 3720
cccuccugac uaaggugguu gaggauggag gaagcauuaa ucuggccucu cauugucaau 3780
ugggccaggc ucucuuacac uauaggcuau uggggucauc aguguccccu gcuuucgauc 3840
aguauuguag cagugccuuc acauccagag acccugccuu agguuacuuc cugauggaca 3900
acccauacgc ugcugggcug cuugguuuua aauacaaccu guggaaugcu ugugugaaua 3960
acacacuagg gaagaaguau aagcagaugu uaaucaaccc ccugagacau aggccucugg 4020
agucaacaac aucaggaacu uuacuucaca gucaucauau ugcuuggggg aaucggaaaa 4080
aaugggaaag gcugguuaga gcuuuggagu uacccgagaa uuggagggau cugauagagg 4140
aggauccagu aauccuauau agaaaggccc aaacagagag agacuuggag cuuagguugg 4200
ccgagaaaau gcacucaccu ggaguuugcu caucucugag uaagggaaau gcugucacua 4260
gaauaauagc uggcucaguc uacauacuga ccaggaaugu cuuaaccauc guuggaggga 4320
gaacaaaggu gaguuugcuc aaagcuauac ucgaagagag ggacgggguu gacccgcucu 4380
ccugggagga agagucccuu cuguuuccga gugcacuaga auaccacuca guugcugcag 4440
cccugagaag acuggacucg agagcagggg uguucaagaa acauaaggaa cgaaggagga 4500
gguccaacau acagauaacc gacucugaug gagacuaccu guuucaacca gaugucauuu 4560
uagccuggag augguuuaac auugacagga ugcacgcugc uguucgggug aaggagagga 4620
uguaucagaa auuaaggcag gcgcugccgu gggugagaga uaccccggag gaaacagugg 4680
cgaacagccc guuugagaau caaauccagc ugcacacguu cuugacuaag acaaccauga 4740
agaggaggga ggugcaucug cucggaacag aagucacuca aagaggcgga aucucaagcc 4800
uaaugacugc cauuuaccag aaccauuucc cuggguucca cuugcaguuc uugaaggaug 4860
aggaggcugc aucacaaucu ucugacuacu caaaacuauc ucacuucauu cacaugacuc 4920
accugucccc auacucugau gacuacaagc agcaguugau ucucaauaga cucgccucua 4980
gcccccagau ugaguucaag gagaaguuug ggaggaguag gaggaacugc cuagccauca 5040
uccagaaguc guugucauug aaugucguag aucuguuaga ccacauaacc uucaguuaua 5100
agaugggagu ugucgggggu uuuguuaaga gacagacaag cacuguuguu aaugggaaag 5160
ugaaguacac uggcguggga acguggguug gauccaugga cggugucaac augcgaauug 5220
ucaucaaugg gacaguggaa uccaauagag ugacuuccau agucguguca aacccucgag 5280
caauguacac gcagaccuuu gggaggcuac ugcagacaug gaugaaggaa auggguguua 5340
cugaagggga cccacacguu aacccaaaug ugauagcuua cuacagaaau gggcacauau 5400
aucagggcgg caggggagga ggagggggcg ucccaauaua ucacucgggg cuaggccuug 5460
agggauuuga uccugccuug gucaaauugg uggauaucca ggugaggaac aacacgguga 5520
gacuuguugc agacuauggc cugaagcacc ucagcaaggu aacuauacug agcuacacca 5580
ccagagacaa ggaugcguca uugaugguga cuaggucuga uuucucagag auaaagccag 5640
cuaugcagua cugguucucu aacacaucau ggccccacga gguagcacag ggagugguga 5700
ccagggcuuc uaagggggaa cucaugccaa auguugauca cagggaguug caauuuugga 5760
ugcaagcacu auuuaagaau gcagcagaga ggaaagggcu ccuugaggag acaauggagc 5820
ucccaauggg gucuaugacu ucgacagugg uuccugaugu guaugaugau uucugggauc 5880
uggucaccuu ugaugagcca caagagcuag aauguuuugg agaggcugug ccuucguggg 5940
aggacuucga cuuugaggug gcuuuuggag gggccaacgu caccaacuau guuccuagug 6000
uccuccgcac ucacccacuu cuagaucaaa ugcucagcag gcugaucagg gacuucuccu 6060
ucggagccau gagcaaguug guuagggaga agguuggaac cccagccaug cuucagucaa 6120
uucaguuguu guccuacuug cugggagugg auucaaauga gauagccauc ccagacuggg 6180
auuuagcguc aaguggcgca gagcaguggg accuuuaaga uguagcaugu ugguucauuu 6240
gauagguuaa uucaaugguu gguggguacu aaaaaaauag uuauguagau ucauuagaac 6300
acacccgguc uuugugu 6317
<210> 2
<211> 2978
<212> RNA
<213> Orientavirus penaei
<400> 2
acacaaagac ggcgcaucaa gugagaauuc aguagugaag uucauuuggu uuagucuuug 60
cgauauguuu guuuuuuuga ucacugcggg cuugcugcca acaaugcugg uuggcaacca 120
acuaucuccc gggaguaugc ucacagcugg ggugggcgaa ugugcccaga guccaccgag 180
auucccauuu gcagacccuu cuaucaaggg ugcaaagaag auacagugga ucaaccuauu 240
ccaucacccc aaacccugcu auuaugaggu uacuuccagc caaagauguu uugaugagcu 300
cggcggaacu ggagcaaggu guaacaacag cagaggaacu gugaugguug augacauaaa 360
cugccaagug gaggcugaag uugggucacu uagugacuca aaggacaucu gugcagugaa 420
uggacauauu cugcacacuu gugcugagac caggacugga cuugaguggg ugaggugggc 480
uuucauguac cgugaaggaa ggaagguccg auuccuagac acguugcaca agagagugaa 540
gcacaaugua gcaggcucag acaguuacac uugcacagga aaucagacag aggggugcaa 600
uggagacuua gccuacugcu cgaacaauga cugugaaguu gccucaaccu gcuucugcac 660
aaccaacaug agagagguga ccucccugau ugucgaugga caagagauag ugccaacaug 720
cuggggagaa ucucugguga ggaucaaucg agagaucaua ggcaauguag gggcauucca 780
gccaugcaca gacugcaagu uccagugcac cgaugagggu gucaagguuu ccacauucau 840
gcaggaagcu gugccuggaa ccauaugcaa gaaccccuau ugcguccaca ucaugguuca 900
guaugaagcu ucaauagucc ugccccugga caugagaguc ucaacugaag agaugacuau 960
cacccuaugg auuaggggca acauugaccc guucgucaac aaaaugacuu gcguguauga 1020
gcaauccugc gcucucauuu ccugccaucu uugccuggag agagcucuca auccacacug 1080
uuucuccugg gcccacuggc uguuagucau gguuuuaauc cuggucagug cagcugccau 1140
ccgcaggauc aucaugcucc ugcucugccu caagugggcg gugauuacau gcuggcacug 1200
ucugaagugg auccaccaaa gggcuucuuc cuugaggagg ggcaaggcua ugcaggacga 1260
ugaccgggcc agauuaacuc aaggacgggc aggagcugcu aacagguuca gggccuaccu 1320
ggccauggcc cugcuguccc ucggucacug cugcuccaca ucagucaccu cuauagcuga 1380
cauccaggac uguucgcaag guacagacgg aguugagacu ugugccuuug accacggagu 1440
ggaucugcau gugaguccaa uuggccagga gaguuguuac uuccucaagg accccaaagg 1500
ggagcacaug gaucaccuuc guauuaagac cgugagcguu cagcugggcu gugaaaagca 1560
gucccuguac uucgucccua gggcaguugg uaagugcgcc aguaucaguc acuguucaac 1620
uguugauggc ugcucaucaa aagcgugccu cgaguucggc aucaaugaca caaaggcuga 1680
cuggacagua gaggcagagc acuacgguug gucuagaugc cguggaaucc caggcugcgc 1740
ugcaaauggg uguuucuacu gugaugaugg gugccucugg uggagagagu acuucaccaa 1800
ccccaagaug gagguguucg agguggucag gugcccgacc uggaucuuua cagugggauu 1860
ggagguugua guguccaacg uuacuacgcc cauaacccua ucuccaggag cuacaaaagc 1920
uguuggcuca gugaggcuga gcuuggaggc ucucagcgug ccaccugagc cauuauuggg 1980
agacugcuuc uauguuaagg acuuggagac aaagauuggg ccuugcaacg agagaggguc 2040
gcugagcccu ggaagaguug gggagcugca augcccaucu aaggagucag caagaagagu 2100
ugacaaaacu uguuuugcga augacgcuau ugucaggucc acugugagcu cggcaggggu 2160
gucaugucac uuuuccuuag uggacccaga gaagguugga accgaccucc cguacaaagg 2220
caauggcuuc acaauccugu cagggaaaga aggaauagug gcucauagua ccuccaccgc 2280
uuugaugucc cucaauauac agcuggggcg cuugcugauc caaaaaagag cugccaggua 2340
caaaugucau gcucaguuug ucaaacugac cggguguuac ucuugcaugg cuggugccac 2400
ccugacuuua uuagugggcu ccaccucuga ugcuucagag gcugugcuca acugcccuga 2460
cgccaacuac accacaauug uuguugcaga caagcuggag aagagcguga ccagcacaau 2520
gcaccugacu gaaucacaga uagacaugaa gugcgagauu guguguccga guagcaggac 2580
auuugucgaa gucuccggua aucucuugua caucccggcc ucggacccug aaacuaggac 2640
caugucggug gacguucaca acaauggagc aucauucaca uggaaccccc ucgggagcua 2700
caagucugcc aucuuguaug gggcaauugc uauagcugca cucauccucc uuucuauucu 2760
ccuuccucuc cuacagaccu cugugaaauc uaagaccaac uaaacgauca uugauuacga 2820
cgggccgaaa ggcucgcauc cgcuaacuaa cuguuuggca gagcuauccg gcucaacaag 2880
ugaggccacu gacauguuga guuucaacgu guggaagauu cuugcuuugc aaucuuaaca 2940
cacuucacua uauuugucau augcgccggu cuuugugu 2978
<210> 3
<211> 1164
<212> RNA
<213> Orientavirus penaei
<400> 3
acacaaagac gagggaucua uagucaauaa cagaguauuc uuaccuuguu caacugacaa 60
cuuuucaaca uggccucuag ugcugagcuu agugaggcug cgaaguucau ucagaacauc 120
gccgucggug aguuuacgau cuucguagcc gacauggaug cguucaucgc agacauccag 180
uuccagggcu uugacccuaa ggucauuguu gcagccuuga ugaagaagga gagcgaugca 240
aacacgcuca aggaggacau cugcaagaug guggccauuc ugugugagag gggaaccaag 300
cucaacaaga ucauggguag gucuucuccg gagggaguuu cucggaucag ggcccucaag 360
gauaaauaug gccuugugga cagugguaag aaggcagaca acaucacccu ugcucggguu 420
gccuucugcc uuccccaguu cacaugcucg uacauggcgg ucugucugaa ccccgcgguu 480
ccauggacca gccuagacga ggggaccuau gccuauccaa ggcccauggc augcagugcc 540
uucgcaaacc ugcuagaggc gaccgauacg gagaugauca acugucaccu guacugggca 600
guccauuuua acaagcugau caacccauca gccaacaagu ccaugaagga aguggcagca 660
gagugccuua aguuugccau gauaggggca aacucgaccc acaucccagc cgcuaagaag 720
aagucaauca gggaagcccu gguccuaucu gcgccaacaa ucaaguucua cucugauaag 780
uucuugagcu ugaccgccua gaaaggcucu cacauuagug gggccagacg gugacacaca 840
aacacacuaa auagacaagc acgacaugag aacaacacag acgagacaca aacaacucac 900
aagacacaca gauacacgca cacauacaca cacacacaca caauccgagg uccugugugu 960
gacuuugaaa ugauggagcu gcacacucac ucgcucuccu ccugcuugug cucaugcuga 1020
ccccccaaug uguggcacau ucaagcauuc ugauugcuug uuccucgguc uuuuauagac 1080
ucaaggagcu cauggucugu ucgucaagaa uauuaugagg caaaccauaa gcauucugau 1140
uguuauaucc cucggucuuu gugu 1164
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> L-12F
<400> 4
ggtgttgtag tgttgcattg gaa 23
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> L-718R
<400> 5
tcatcctcct catggaaaga tactc 25
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> L-496F
<400> 6
cttatgacta tgagctgagg gagag 25
<210> 7
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> L-1460R
<400> 7
gacattcagc ttattactat cgagga 26
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> L-1274F
<400> 8
tcacagagtg gacatagcct tagaa 25
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> L-2342R
<400> 9
acataagtac cctacatagt ttaggttcac 30
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> L-2613F
<400> 10
acgctgaaag cttcctcgaa 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3509R
<400> 11
tgactcaagt gctgcctcag 20
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> L-2688F
<400> 12
gccatccaag gagtgagaga c 21
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3011R
<400> 13
acttcctagc atctgcgctc 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3030R
<400> 14
acttcctagc atctgcgctc 20
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3085F
<400> 15
tttgaggtac atgaaatacg tgtgtt 26
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3108R
<400> 16
ctatgaaacc atgtatgtaa ctag 24
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> L-2942F
<400> 17
gttcaagctg cctgaggaac at 22
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3970R
<400> 18
cctagtgtgt tattcacaca agcatt 26
<210> 19
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> L-3836F
<400> 19
cgatcagtat tgtagcagtg cctt 24
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> L-4677R
<400> 20
ctgtttcctc cggggtatct c 21
<210> 21
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> L-4520F
<400> 21
cgactctgat ggagactacc tgtt 24
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> L-5439R
<400> 22
atattgggac gccccctc 18
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> L-5317F
<400> 23
catggatgaa ggaaatgggt g 21
<210> 24
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> L-6308R
<400> 24
ccgggtgtgt tctaatgaat ctac 24
<210> 25
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> L-6104F
<400> 25
agccatgctt cagtcaattc agt 23
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> L-9R
<400> 26
agatgccctt gttaccccga 20
<210> 27
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> M-90F
<400> 27
gcttgctgcc aacaatgct 19
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> M-1090R
<400> 28
ccaggagaaa cagtgtggat tg 22
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> M-966F
<400> 29
tatggattag gggcaacatt gac 23
<210> 30
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> M-2007R
<400> 30
tccaagtcct taacatagaa gcagtc 26
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> M-1844F
<400> 31
atctttacag tgggattgga ggtt 24
<210> 32
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> M-2818R
<400> 32
gtaatcaatg atcgtttagt tggtctt 27
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> M-2692F
<400> 33
cgggagctac aagtctgcca t 21
<210> 34
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> M-252R
<400> 34
ttggggtgat ggaataggtt g 21
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> S-380F
<400> 35
acagtggtaa gaaggcagac aac 23
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> S-1120R
<400> 36
cctcataata ttcttgacga a 21
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> S-644F
<400> 37
tgaaggaagt ggcagcagag t 21
<210> 38
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> S-1145R
<400> 38
taacaatcag aatgcttatg gtttg 25
<210> 39
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> S-903F
<400> 39
gacacacaga tacacgcaca catac 25
<210> 40
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> S-532R
<400> 40
atgccatggg ccttggata 19
Claims (10)
1. one kind based on prawn east virus (Orientavirus penaei) genome sequence detection method and its application,
It is characterized in that, the deposit number of the virus is CCTCC No. V201864.
2. detection method according to claim 1, which is characterized in that its genome sequence such as SEQ ID No 1, SEQ ID
Shown in No 2 and SEQ ID No 3.
3. detection method according to claim 1, which is characterized in that the specific sequence of the detection method be
(a): the RNA sequence of SEQ ID No 1 or SEQ ID No 2 or SEQ ID No 3；Or
(b) :(a) the DNA sequence dna of conversion；Or
(c) :(a) and (b) >=70% homologous RNA or DNA sequence dna；Or
(d): RNA or DNA sequence dna comprising (a) and (b) or degeneracy base (c)；Or
(e): the RNA or DNA sequence dna of the degeneracy base of (d) that is replaced with hypoxanthine or other equivalent bases；Or
(f): with (a) or (b) or (c) or (d) or (e) complementary RNA and DNA sequence dna；
Within the scope of 1 or 1 or more 12nt-3000nt segment.
4. detection method according to claim 3, which is characterized in that this method includes at least 1 pair from specific sequence
It selects the single stranded DNA of 12nt-30nt or RNA segment is specific primer.
5. detection method according to claim 4, which is characterized in that the specific primer is selected from SEQ ID No
Specificity in sequence shown in 4-SEQ ID No 40 and SEQ ID No 4-SEQ ID No 40 between any 2 sequences
Segment in sequence.
6. detection technique according to claim 3, which is characterized in that the detection technique can also include 1 from special
The polynucleotide sequence segment of the 15nt or more selected in property sequence is specific probe.
7. according to detection technique described in claim 5 and 6, which is characterized in that the primer and probe uses polymerase chain
The isothermal duplication or other nucleic acid amplifications or nucleic acid hybridisationdetection technology of formula reaction or loop-mediated isothermal amplification or dependence unwindase.
8. detection technique according to claim 3, which is characterized in that determine the detection point of the specificity of the detection technique
Son is
(a): the polypeptide of the complementary series translation of the nucleic acid sequence of SEQ ID No 1 or SEQ ID No 2 or SEQ ID No 3
It is all or part of；Or
(b): having all or part of >=70% homologous polypeptide sequence with (a)；Or
(c) :(a) or antibody (b)；Or
(d) :(a) or aptamer (b).
9. detection technique according to claim 8, which is characterized in that using polyclonal antibody, monoclonal antibody, single-stranded anti-
Enzyme Linked Immunoadsorbent Assay, fluoroimmunoassay, immuno-chromatographic test paper strip or the aptamer that body or aptamer are established
The specific amplification of sequence or other immunologys or quasi- immunology detection.
10. the application of detection method as described in claim 1, which is characterized in that will test every reagent needed for method and match
It is set to matched kit or corollary equipment, the detection for prawn east virus or other homologous viruses.
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| CN110866893A (en) * | 2019-09-30 | 2020-03-06 | 中国科学院计算技术研究所 | Pathological image-based TMB classification method and system and TMB analysis device |
| CN111019909A (en) * | 2019-12-27 | 2020-04-17 | 中国水产科学研究院黄海水产研究所 | Crustacean flavivirus and detection method and application thereof |
| CN116376848A (en) * | 2022-12-13 | 2023-07-04 | 中国水产科学研究院黄海水产研究所 | Artemia virus and detection method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110866893A (en) * | 2019-09-30 | 2020-03-06 | 中国科学院计算技术研究所 | Pathological image-based TMB classification method and system and TMB analysis device |
| CN110866893B (en) * | 2019-09-30 | 2021-04-06 | 中国科学院计算技术研究所 | Pathological image-based TMB classification method and system and TMB analysis device |
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| CN111019909A (en) * | 2019-12-27 | 2020-04-17 | 中国水产科学研究院黄海水产研究所 | Crustacean flavivirus and detection method and application thereof |
| CN116376848A (en) * | 2022-12-13 | 2023-07-04 | 中国水产科学研究院黄海水产研究所 | Artemia virus and detection method and application thereof |
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| Publication number | Publication date |
|---|---|
| CN109628638B (en) | 2019-12-10 |
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Application publication date: 20190416 Assignee: Dongying Kuohai Products Technology Co.,Ltd. Assignor: YELLOW SEA FISHERIES Research Institute CHINESE ACADEMY OF FISHERY SCIENCES Contract record no.: X2023370010032 Denomination of invention: Detection method and application based on shrimp oriental virus genome sequence Granted publication date: 20191210 License type: Common License Record date: 20231219 |