CN108315337A - Wheat Yellow strip virus and its NASH detection methods, detection kit - Google Patents
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Abstract
The present invention relates to a kind of Wheat Yellow strip virus and its NASH detection methods, detection kits.Present invention finds a kind of new virus, that is, Wheat Yellow strip virus WYSV, its full length sequence is as shown in SEQ ID NO.3, and devise shown in the primer pair SEQ ID NO.1/2 of a pair of of specific amplification WYSV, prepare DNA probe, detection WYSV viruses by marker of digoxin.The present invention also provides a kind of WYSV virus agent boxes of NASH detections, which includes：Probe PCR reaction solution, NASH reaction reagents, nitrocellulose filter, hybridization bag, WYSV positive control samples and negative control sample.The detection method and detection kit can a batch detection sample, can detect to low-cost high-efficiency WYSV viruses in a short time, testing result is reliable, easy to operation, high specificity, high sensitivity, and time-consuming short, application prospect is good.
Description
Technical field
The invention belongs to agrobiology technical fields, and Wheat Yellow strip virus (Wheat is used for more particularly to one kind
Yellowstriate virus, WYSV) and its NASH detection methods, detection kit, it can reach quickly, sensitively, and conveniently examine
Survey the purpose of WYSV in plant body.
Background technology
Wheat is one of staple food crop all over the world.Virosis is to endanger the important disease of one kind of Wheat Production,
The virus causing disease reported is up to more than 50 kinds.Virus can cause the wheat plant infected to generate leaf chlorosis, plant is downgraded, effectively
Serious symptoms, the influences to wheat growth and yield such as tiller reduction are huge.The virus that many infects wheat is special by mediator insect
Heterosexual transmission, including aphid, small brown rice planthopper and leafhopper etc..With global warming, no-tillage and high-toxic pesticide disabling etc. because
Element makes biography virus mediator insect populations be gradually increased, and variety resistance degree is generally more low in addition so that worm passes wheat crops disease
Viral disease China harm is on the rise, occurring area and the degree that causes harm increase rapidly, become influence grain security an important factor for
One of.In recent years, endangering serious arbovirus disease in our most of area of wheat has yellow stunt of wheat, wheat rosette stunt, wheat
Dwarf wilt and the green short disease of wheat etc. are a variety of, and wherein the cause of disease of yellow stunt of wheat is luteovirus (Barley yellow
Dwarf virus, BYDV), it is specifically spread through sex intercourse by wheat aphid propagation.Wheat rosette stunt is by northern wheat mosaic poison (Northern
Cereal mosaic virus, NCMV) cause, the green short disease of wheat is by rice black-streaked dwarf virus (Rice black-
Streaked dwarf virus, RBSDV) cause, both viruses are by small brown rice planthopper (Laodelphax striatellus
Fallen it) propagates.The cause of disease of wheat dwarf wilt is wheat dwarf virus (Wheat dwarf virus, WDV), mainly passes through Jie
Body item sand leafhopper (Psammotettix striatus L.) is propagated in a manner of persistently walking around to.And barley Huang item point mosaic disease is
The another kind virus propagated by small brown rice planthopper found on China's wheat in recent years, cause of disease is barley Huang item point mosaic virus
(Barley yellow striate mosaic virus, BYSMV), is a kind of cytoplasm rhabdovirus.
The present inventor in 2016 is found that a kind of new Disease symptoms when Hancheng Region, Shaanxi carries out disease field investigation, and small
The symptom that wheat dwarf wilt viral disease generates is different, and the wheat infected is downgraded without apparent, shows as serious yellow, incidence of leaf along leaf
Arteries and veins chlorosis is gradually developed into and is dried up since blade tip, last withered (Fig. 1).It has been observed that the morbidity main agricultural of field youngster
Pest is different husky leafhopper (Psammotettix alienus Linnaeus), and different husky leafhopper is caught back raising and is planted to healthy wheat
In strain, there is illness similar with field in wheat plant after 3 weeks, and the preliminary proof virosis is propagated by different husky leafhopper,
It is likely to a kind of also undocumented new virus.It is therefore desirable to identify the virus, detection.
Detection method applied to plant virus usually has the technological means such as biology, serology and molecular biology.But
For new virus, since it is propagated with two kinds of viruses of WDV by different husky leafhopper, there are similarity, biology mirror in symptom again
Surely it can not complete accurately to differentiate；Serology have many advantages, such as it is fast and convenient, high-throughput, depend on sero-fast virus detection techniques
It is most ripe with elisa technique, it is most widely used.But due to being limited by antiserum preparation is horizontal, the country there is no commercialization
Antiserum；The advantages that molecular detection technology is quick, sensitive, special, accurate because of its is widely used in the quick inspection of plant virus
In survey and the early screening and diagnosis of identification and disease.PCR methods are more common, but these experimental techniques are not easy to grasp, and
It is influenced by cost problems such as reagent and expensive equipments, it is difficult to be promoted in plant protection department of base.Based on viral known nucleic acid sequence
The spot hybridization test (nucleic acid spot hybridization, NASH) to grow up has high specificity, sensitive
Spend the features such as high, which is the principle that can be combined with each other according to complementary single nucleic acid strands, by one section of viral nucleic acid it is single-stranded with
Certain mode is marked, and is made probe, then is hybridized with complementary sample to be tested nucleic acid, by the hybrid nucleic acid with probe come
Indicate the presence of cause of disease.Especially with nonradioactive probe marker such as digoxin (DIG), have many advantages, such as it is safe and stable,
It is widely used in the detections of various plants virosis such as yellow stunt of wheat, cucumber green mottle virosis.The present invention is profit
Developed for the WYSV viruses carried in wheat disease plant body with principles above applied suitable for grass-roots unit it is special, efficient
Nucleic acid spot hybridization detect box.
Invention content
The object of the present invention is to provide a kind of new Wheat Yellow strip virus (Wheat yellow striate virus,
WYSV), which is carried by different husky leafhopper, is infected wheat and is downgraded without apparent, shows as serious yellow, incidence of leaf along vein
Chlorosis is gradually developed into and is dried up since blade tip, last withered.
The nucleic acid spot hybridization detection technique of digoxigenin labeled is introduced into the detection of new virus WYSV by the present invention, first
Network analysis is carried out to the nucleotide sequence of WYSV viruses and its allied species, devises the specificity for specific detection WYSV
Primer is cloned into carrier after obtaining special DNA in genome using PCR methods and obtains recombinant plasmid, then again with
It is template, and the DIG-dNTPs of use carries out PCR amplification, obtains the DNA probe of the WYSV Partial Fragments of digoxigenin labeled, exempt from
Epidemic disease testing result shows：The WYSV probes of preparation have stronger specificity and high sensitivity, establish WYSV-NASH detection skills
Art.The detection of the doubtful WYSV standard specimens in field is applied successfully to based on the NASH detection techniques that this probe is established.Quickly to examine
Disconnected, detection WYSV provides a kind of new way effectively, safe.
The present invention also provides the detection kits.
Wheat Yellow strip virus (Wheat yellow striate virus, WYSV), encodes the nucleosides of the viral gene
Sour full length sequence is as shown in SEQ ID NO.3.
A kind of nucleic acid spot hybridization detection method for WYSV Wheat Virus, in the PCR system reaction solution for preparing probe
In, include following a pair of of specific primer pair, nucleotides sequence is classified as：
SEQ ID NO.1：5’ggtgctcagtgtcgctttct-3’；
SEQ ID NO.2：5’acctgctgttccattcttgtc-3’.
The probe is to pass through DNA probe made from PCR methods by marker of digoxin.
The 25 μ L of PCR system reaction solution total volume, wherein containing 0.5 μ L recombinant plasmids, 2 a concentration of 2.5mM of μ L
DIG-dNTPs, 0.2 μ L rTaq and the above-mentioned specific primers of a concentration of 10 μm of ol/L WYSV are to each 0.5 μ L, the recombination
Plasmid is the pMD-18T recombinant vectors of the target fragment of portion gene containing WYSV.
The recombinant vector is the PCR product to being obtained with reverse transcription PCR amplification by the above-mentioned specific primers of WYSV,
It is connected to after purification on pMD-18T carriers, in conversion to 5 α E. coli competents of DH.
The pcr amplification reaction condition is：94 DEG C of 3min, 94 DEG C of 30s, 56 DEG C of 45s and 72 DEG C of 90s are recycled for 35 totally, and 72
℃10min。
The above method is detecting the application in susceptible crop plant body in WYSV viruses.
The crops are wheat, barley or oat.
A kind of kit for detecting WYSV viruses, the kit include：PCR system reaction solution, NASH reaction examinations
Agent, nitrocellulose filter and hybridization bag；The NASH reaction reagents are composed of the following components：
Contain above-mentioned specific primer pair in the PCR system reaction solution.
Further include WYSV positive control samples and negative control sample in the kit.
The present inventor in 2016 is found that a kind of new Disease symptoms when Hancheng Region, Shaanxi carries out disease field investigation, and small
The symptom that wheat dwarf wilt viral disease generates is different, and the wheat infected is downgraded without apparent, shows as serious yellow, incidence of leaf along leaf
Arteries and veins chlorosis is gradually developed into and is dried up since blade tip, last withered (Fig. 1).It has been observed that the morbidity main agricultural of field youngster
Pest is different husky leafhopper, different husky leafhopper is caught back raising to healthy wheat plant, wheat plant occurs and field phase after 3 weeks
As illness, the preliminary proof virosis is propagated by different husky leafhopper.
According to the viral nomenclature of host plus symptom rule, new virus is fixed tentatively into entitled Wheat Yellow strip virus (Wheat
yellow striate virus,WYSV).Pass through transcript profile sequencing analysis, the determination and analysis of sequence to susceptible wheat samples
It discloses：The virus is negative adopted single stranded RNA, and entire WYSV full-length genomes are 14,486nt, as shown in SEQ ID NO.3；Gene
Group sequence is respectively provided with 58.1% and 57.7% nucleotides sequence with two rice yellow dwarf virus strain RTYV and RYSV genomes
Row consistency.Identical as rice yellow dwarf virus, WYSV contains 7 open reading frame (ORF), with " N-P-P3- on antisense strand
M-P6-G-L " sequences encode albumen successively.In addition, WYSV genomes have 76nt 3' end targeting sequencings and the ends 258nt 5' to trail
Sequence, ORF are separated by conservative intergenic sequence.Amino acid (aa) sequence identity highest between the L albumen of WYSV and RTYV,
It is 63.8%, but minimum with P6 albumen consistency, only 29.5%.Phylogenetic Analysis shows WYSV and plant rhabdoviruses
(Nucleorhabdovirus) member is classified as cluster.Simultaneously using transmission electron microscope observing also in susceptible wheat leaf blade tissue
In be found that the virion of a large amount of bullet shaped.Through indoor biological inoculation, it has also been found that, which can be passed by different husky leafhopper
It is multicast on barley and oat.Therefore, WYSV is a kind of thin on the gramineous crops such as wheat by being infected with the different husky leafhopper of poison
Karyon rhabdovirus.In addition the present inventors have additionally discovered that after different sand leafhopper carrying WYSV, then inoculation WDV is carried out, the wheat infected
WDV detects ratio and declines in group, WYSV recall rate highers.That is this nucleus rhabdovirus is in the different husky leafhopper of mediator
Internal massive duplication causes the ratio of outflow higher, thus it is speculated that the risk of this new virus prevalences of WYSV is very high.It is therefore desirable to
It is monitored early warning before the big generation of the disease, efficiently quick detection method is established, to carrying the host plant of the virus
And mediator is detected, and is played an important role to preventing the virosis.
Nucleic acid Dot hybridization technology of the present invention is the principle that can be combined with each other according to complementary single nucleic acid strands,
It is marked in some way by one section of viral nucleic acid is single-stranded, probe is made, then hybridize with complementary sample to be tested nucleic acid, passes through
Hybrid nucleic acid with probe indicates the presence of cause of disease.Nucleic acid hybridization technique based on nucleic acid sequence, can more acurrate spirit
The type of virus is identified quickly.The nucleic acid spot hybridization detection technique of digoxigenin labeled is introduced the detection of WYSV by invention, is fast
Speed diagnosis, detection WYSV are provided a method.Specific invention content is 1) the different husky leafhopper of mediator and WYSV poison source sample
Preparation；2) identification of Wheat Yellow strip virus (WYSV)；3) extraction of plant total serum IgE；4) design of specific primer and contain
The acquisition of the recombinant plasmid of target fragment；5) preparation of digoxin labelled probe；6) foundation of NASH detection methods；7) side NASH
The specific detection of method；8) sensitivity technique of NASH methods；9) NASH is applied to the detection of the doubtful WYSV samples in field.This hair
The bright nucleotide sequence to WYSV viruses and its allied species carries out network analysis, devises for the special of specific detection WYSV
Property primer, the DNA probe of digoxigenin labeled has been obtained using PCR methods, immune detection the result shows that：The DNA of the WYSV of preparation is visited
Needle set has stronger specificity and high sensitivity, establishes WYSV-NASH detection techniques.
Above-described specific primer can be used as detection applied to this viruses of WYSV and prepared by DNA probe answers
With.
Hancheng Region, Shaanxi is picked up from the WYSV poison source, positive plant is accredited as through high-flux sequence identification and PCR, through being situated between
The different husky leafhopper live body feeding of body is stored in susceptible wheat breed and raises wheat No. 12.
The different husky leafhopper population picks up from Hancheng Region, Shaanxi, is raised on wheat seedling throughout the year.
The mediator raising and host's wheat lines as feed are planted in 22 DEG C of incubators, 16h illumination, 8h
Dark, intensity of illumination 20000Lx.Leafhopper is transferred on the seedling of novel species by every four weeks, preserves mediator in this approach.
The present invention has the following advantages：
1. the present invention use nonradioactive probe marker such as digoxin, "dead" pollution, it is safer the advantages that.
2. the present invention is more stable with probe prepared by digoxin, storage time is at least 1 year, and the hybridization containing probe
Buffer solution can Reusability 4~5 times, saved testing cost.
3. the reaction systems ingredients such as digoxigenin-probe, hybridization buffer and the cleaning solution of the invention prepared are assembled into reagent
It is simple to operate without the instrument of any costlinesses such as PCR, enzyme-linked analyzer after box, it is very applicable to grass-roots unit, it takes into account
Practicability and rapidity, have a good application prospect.
Description of the drawings
The symptom figure of Fig. 1 .WYSV virus infection wheats.
Fig. 2 .WYSV infect the symptom of gramineous crop through artificial infection, and host is respectively (A) wheat；(B) barley；(C)
Oat.
The transmission electron microscope photo of Fig. 3 .WYSV virions.
The virus genomic analysis of Fig. 4 .WYSV.(A) viral genome structure schematic diagram；(B) it is indicated respectively with (C)
Fluctuation distribution is presented in the reading sequence that wheat and leafhopper polypide obtain through high-flux sequence on virus genome RNA.
The genome sequence feature of Fig. 5 .WYSV.(A) complementary series of genome 3 ' and 5 ' ends；(B) each of virus
The conserved sequence of ORF spacer regions.
WYSVs and the other bullet shapes that infect plant of Fig. 6 according to L protein amino acid sequences using MEGA7.0 software buildings
The systematic evolution tree of virus.The biography virus mediator and spacer region conserved sequence of the corresponding each virus in figure right side.
PCR amplification result prepared by Fig. 7 digoxigenin-probes；Wherein：M represents DL2000DNA Marker, from top to bottom according to
Secondary is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp；1 swimming lane be WYSV virus specific primers with dNTPs just
The result often expanded.2 swimming lanes are the result that WYSV viral samples specific primer is expanded with DIG-dNTPs.
The testing result of Fig. 8 .NASH method specificity；Wherein, 1 is healthy wheat RNA；2~5 be followed successively by WYSV, WDV,
The diseased plant RNA of BYDV-GAV, BYDV-PAV, WYDV-GPV and BYSMV.
The testing result of Fig. 9 .NASH method sensitivity；(A) it is No. 1 sample, (B) is No. 2 samples；Wherein 1 is sample RNA
Stoste, 2~7 are followed successively by RNA stostes through 10-1~10-6Dilution.
Figure 10 .NASH methods are applied to the testing result of crop field sample.
Specific implementation mode
Present invention will be further explained below with reference to specific examples, these embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition.
It needs it is emphasized that although what the present invention illustrated is mirror for susceptible wheat plant vivo detection WYSV viruses
Determine technology, but WYSV is to carry a variety of wheat germ plasm resources such as virus infection wheat, barley, oat by different husky leafhopper, therefore answer
With the present invention to the wheats germ plasm resources such as any host such as wheat, barley, oat for causing WYSV viruses and the different yarn leaf of mediator
The detection of cicada is also included within interest field of the presently claimed invention.
Reagent raw material described in following embodiments is commercially available common raw material, reagent is matched in addition to especially indicating source
System is all made of conventional method.Biomaterial is preserved by the laboratory of the applicant, can be with external disclosure granting.
The preparation of the different husky leafhopper of 1. mediator of embodiment and WYSV poison source sample
The source of the different husky leafhopper of 1.1 mediators and preservation
Different sand leafhopper acquisition is from the common field of Hancheng Region, Shaanxi, and button cover is raised on healthy wheat after purification for detoxification.Raise item
Part is：22 ± 1 DEG C, in the illumination box of 20000lx conditions.
The source of 1.2 Wheat Yellow strip virus (WYSV) and preservation
In April, 2016 is found that a kind of new Disease symptoms when Hancheng Region, Shaanxi carries out disease field investigation, short with wheat
The symptom that contracting virosis generates is different, and the wheat infected is downgraded without apparent, shows as serious yellow, incidence of leaf loses along vein
It is green, it gradually develops into and dries up since blade tip, it is last withered.It acquires 2000 head lobe cicada of idiopathy field and preserves to greenhouse
In insect prevention cylinder mould, it is inoculated into the raising on wheat 12 of a leaf phase through insect, is transferred to 22 ± 1 DEG C, the illumination cultivation of 20 000lx conditions
It is grown in case.3 weeks rear blades have the plant of yellow striped symptom to breed malicious source as inoculum, carry out next step verification.
It takes in 60 adult leafhopper feedings to Symptomatic plant, insect, which is inoculated into a basin health wheat, after 72 hours plants
In strain, after 4 days, it is transferred to by 3 heads/plant on new healthy tree.72 hours seed stages remove leafhopper later, and inoculation plant is placed on ring
It is grown in the case of border.Corresponding nontoxic leafhopper is as parallel control.
The identification of 2. Wheat Yellow strip virus (WYSV) of embodiment
2.1 Biology identification
Mediator in order to detect virus passes poison specificity, selects nontoxic brown paddy plant hopper (Nilaparvata lugens), ash
Three kinds of plant hoppers of plant hopper (Laodelphax striatellus) and white backed planthopper (Sogatella furcifera Horv á th) with
And grain aphid (Sitobion avenae), green bugs (Schizaphis gramienum) and rhopalosiphum padi
(Rhopalosiphum padi) carries out passing malicious experiment as experimental insect.In addition barley (kind name is also had chosen：Imperial beer wheat 3
Number) and oat (kind name：The black oat of bank) do preliminary host range identification.The malicious step of feeding poison and biography tested above is the same as implementation
Example 1.2, all detection plant, which are placed in illumination box, to be grown, and disease symptom is observed after 2-3 weeks.Biological experiment result
Show that WYSV can be traveled to effectively on the gramineous crops such as barley and oat (Fig. 2) by leafhopper, after being inoculated with 3 weeks, barley
Golden yellow is presented in the blade of kind dragon beer wheat No. 3, and the blade of the black oat of bank shows as peony.The specificity experiments of mediator
Show that WYSV is only capable of specifically spreading through sex intercourse by different husky leafhopper, and cannot be long by brown paddy plant hopper, small brown rice planthopper and white backed planthopper and wheat
Pipe aphid, green bugs and rhopalosiphum padi are propagated, and very strong mediator virus-specific is shown.
2.2 Electronic Speculum are observed
WYSV diseased plant blades with classical symptom are cut into small pieces in throwing to glutaraldehyde fixer, then again with 1% osmium
Acid is fixed after carrying out, after dehydration, replacing, being impregnated with series of steps, using epoxy resin CY212 (Agar Scientific,
Standsted, UK) it is used as embedding medium, tissue specimen is embedded in resin.By ultra-thin section 5%W/V acetic acid uranium and 2%
W/V lead citrates (PH12) carry out double dyeing, with H-7500 types transmission electron microscope (Hitachi High-Technologies, day
This) it is observed.It has been observed that virus is gathered in the vascular tissue of disease plant blade, in the cell of susceptible tissue
The cell nuclear bomb shape disease for infecting plant that the virion of a large amount of bullet shapeds, structure and form have been reported with other is found that in core
It is very similar that poison belongs to virus.The typical virions of WYSV are about 180-210nm × 35-40nm, and cluster is arranged in parallel
In the perinuclear space expanded (Fig. 3).
2.3 high-flux sequences obtain the nearly full length sequence of WYSV viral genomes
With the total serum IgE of sick leaf and toxic leafhopper sample of the TRIzol methods extraction with classical symptom, two sets are established respectively
The ribosomal libraries RNA-seq are gone to, using Illumina HiSeq X-ten microarray datasets, sequencing reading length is 2 × 150bp (texts
Library, which builds and is sequenced, to be executed by Beijing shellfish is auspicious with health biology information technology Co., Ltd).From two texts of sick leaf and toxic leafhopper
The original reading sequences (read) of 2 × 150bp for obtaining 103,396,270 and 61,864,662 in library respectively carry out initial data
Data filtering, removes joint sequence therein and low quality reads sequence, obtains the sequence of high quality.De nove cluster assemblings are carried out,
The 466,904 of 259,179 contigs (contigs) and 200-28,405nt that length is 200-19993nt are respectively obtained
A contigs.Use BLASTx programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in ncbi database
Middle comparison finds, infected leaves and with two that the length received respectively in malicious leafhopper transcript profile is 14512nt and 14399nt compared with
Long contig can be matched on multiple albumen of known Rhabdoviral genes group, and such as rdrp gene, but sequence is consistent
Property be less than 65%, thus it is speculated that the virus be a completely new plant rhabdovirus.
The genome structure and signature analysis of 2.4WYSV
According to two contig (14512nt and 14399nt) design primer that high-flux sequence obtains, using 3 ' and 5 '
The amplification of RACE kits obtains the end sequence of virus, and the WYSV full-length genome sequences that length is 14486nt have been obtained through splicing
It arranges (SEQ ID NO.3), shows that the sequence of gained is accurate through the verification of RT-PCR cDNA clones.Blast is analysis shows the disease
Poison equally, for negative adopted single strand RNA virus, encodes 7 altogether with rice yellow dwarf virus (Rice yellow stunt virus, RYSV)
A open reading frame (Open Reading Frame, ORF).Each ORF corresponds to the albumen encoded：Nucleocapsid protein N
(ORF1), phosphoprotein P (ORF2), it is assumed that albumen P3 (ORF3), matrix protein (ORF4), glycoprotein G (ORF5), it is assumed that albumen
Seven albumen of P6 (ORF6) and polymerase protein L (ORF7), protein characteristic are as shown in table 1.As shown in Figure 4 A, WYSV genomes are false
Fixed each sequence in the gene sequence is followed successively by 3'-leader-N-P-P3-M-G-P6-L-trailer-5'.By wheat leaf blade and leafhopper
The obtained reading sequence of high-flux sequence compare again in WYSV full-length genomes, both find on the geneome RNA of virus
Similar distribution is showed, and the corresponding reading sequence in each regions ORF has more very than the reading ordinal number amount of adjacent noncoding region
More (Fig. 4 B, 4C).
The protein specificity of 1 WYSV genome encodings of table
Note：Nt, nucleotide；Aa, amino acid；KDa, kilodalton.
It is (leading that the further sequence analysis of virus finds that the 3 ' leader that length is 76nt are contained at the both ends RNA of virus
Sequence) and 258nt 5 ' trailer (tailer sequence).Rear 9 nt (5 '-of the trailer tailer sequences at the ends 5'
UGGUGGUGU-3 ') with 3 ' end targeting sequencings before 9 sequence (5 '-ACACCACCA-3 ') complete complementaries, formed rhabdovirus sequence
Typical short panhandle shape structure (Fig. 5 A) in row, and 9 sequences and rice yellow dwarf virus RYSV are completely the same.In 3 ' leader
Find that there are a 4 nucleotide UGUU motifs (44-47) in region sequence, this is one conservative feature of minus-stranded rna virus.
Genetic interval sequence between plant rhabdovirus genome is all highly conserved between gene of each virus
, each ORF of WYSV genomes is separated by highly conserved 3 '-UAUAAAUUUUUGGGGUUG-5 ' of genetic interval sequence
(3'/N spacer regions exception), this sequence and other plant rhabdovirus are similar (Fig. 5 B).Between known Rhabdoviral genes
Septal area is made of three parts, poly (U) tail (element I) of the ends 3', the short non-transcribed element of an each gene of segmentation
(element II) and the Conserved Elements (element III) for being located at each subsequent gene beginning.With other plant rhabdovirus phase
Than, the element I of WYSV is similar with the sequence of element II, and wherein element II is there are four G residues, than RYSV, MMV, TaVcV,
The more G of the nucleus such as PYDV and EMDV rhabdovirus.And many cells such as the UUG and RYSV of element III, PYDV and SYNV
Core rhabdovirus is consistent.
It is carried out based on 20 kinds of rhabdoviruses (WYSV and 19 representative member) for infecting plant of L protein amino acid sequences pair
Phylogenetic Analysis, phylogenetic tree are shown：Two strain RTYV of WYSV and nucleus Rhabdovirus rice yellow dwarf virus
It is gathered in an independent branch, is gathered around there are one common ancestors (Fig. 6) with RYSV.
The extraction of 3. plant total serum IgE of embodiment
Extract the total serum IgE of wheat samples to be detected respectively using TRIzol (Invitrogen, USA) method.Specifically extracted
Cheng Wei：By 0.1g it is fresh or frost sample, be put into 1.5mL centrifuge tubes, be transferred in liquid nitrogen freeze rapidly, be fully ground to
It is powdered, 1mLTRIzol is then added, acutely rocks mixing, stands 5min on ice；200 μ L chloroforms are added, firmly shake 15s, ice
Upper standing 5min.4 DEG C, 12,000rpm centrifugation 10min；Supernatant is transferred to new centrifuge tube, is added and the isometric isopropyl of supernatant
Alcohol gently overturns mixing, stands 10min on ice；4 DEG C, 12,000rpm centrifugation 15min remove supernatant；It is added the 75% of 1mL precoolings
Ethyl alcohol (the DEPC water of sterilizing is prepared), washing precipitation.4 DEG C, 12,000rpm centrifugation 5min abandon supernatant, repeat above-mentioned washing step,
With thorough wash clean salinity；Supernatant is sucked out as possible with pipette tips.It is opening up in ventilating kitchen, it is gone out with 40 μ L after dry 1-2min
The DEPC water dissolutions of bacterium.1 μ L RNA are taken to measure the purity and concentration of RNA sample with NanoDrop-2000 ultraviolet specrophotometers
Value.Qualified samples are placed in -70 DEG C of refrigerators and preserve for use.
The design of 4. specific primer of embodiment and the acquisition of the recombinant plasmid containing target fragment
It is soft using Primer Premier 6.0 according to the WYSV whole genome sequences overall length (SEQ ID NO.3) of measurement
Part designs the specific detection primer of this virus, and specific primer is SEQ ID NO.1 and SEQ ID NO.2, by upper marine growth
Engineering Co., Ltd synthesizes.
CDNA is synthesized using primed reverse transcription.CDNA synthetic system total volumes be 20 μ L, wherein RNA (about 1 μ g) 3 μ L,
1 μ L, DEPC ddH of downstream primer (SEQ ID NO.2) (10 μm of ol/L)2O complements to 10 μ L, and 2-3min are denaturalized for 90 DEG C after mixing,
It is immediately placed on 2min on ice；Then 4 μ L, M-MLV reversions of dNTP Mix (2.5mM each) 2 μ L, 5 × MMLV-buffer are added
Record 0.5 μ L, DEPC ddH of enzyme (Promega, USA) 1 μ L, RRI (Takara, China)2O complements to 10 μ L.After mixing, 37 DEG C
It is incubated 1h, 70 DEG C of 10min, -20 DEG C of preservations.
25 μ L of total volume in PCR system, wherein dNTPmix, 0.2 μ L containing 2 μ L above-mentioned cDNA, 2 a concentration of 2.5mM of μ L
RTaq, 2.5 μ 10 × PCR of L buffer and a concentration of each 0.5 μ L of 10 μm of ol/L WYSV upstream and downstream primers.By following amplification
Program carries out：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 56 DEG C of renaturation 45s；72 DEG C of extension 1min, 35 cycles；Last 72
DEG C extend 10min, saves backup at 4 DEG C.The band (see Fig. 7 swimming lanes 1) that size is about 1200bp is obtained through PCR amplification, with
It is expected that gene target fragment size (1193bp) is almost the same.Purified PCR product is connected to pMD-18T carriers
(TaKaRa) it on, is transformed into 5 α E. coli competents of DH.It is identified through PCR and chooses 3 positive colonies by giving birth to work bioengineering
(Shanghai) limited liability company completes sequencing, determines the recombinant plasmid for obtaining the segment of Gene Partial containing WYSV.
The preparation of 5. digoxin labelled probe of embodiment
DIG probes label uses PCR methods, in kit (DIG PCR probe synthesis kit, Roche)
DIG-dNTPs (DIG-11-dUTP containing 0.7mmol/L, 1.3mmol/L dTTP) replaces the dNTPmix in the normal systems of PCR,
The 0.5 μ L of recombinant plasmid obtained using embodiment 4 is templates, other PCR reagent components unchangeds, carry out PCR amplification, and amplification program is same
Embodiment 4.1% agarose gel electrophoresis is carried out after reaction, because the molecular weight of DIG is big, can be seen that from Fig. 7 swimming lanes 2
Marked band obviously lags behind common unlabelled band, illustrates that this probe marks successfully.
The foundation of embodiment 6.NASH detection methods
6.1 point samples with hybridize
Take the total serum IgE sample spot of 1 μ L in nitrocellulose nylon membrane (Amersham HybondTM-N+, GE
Healthcare on), film is placed in UV UV crosslinking instrument fixed；The nitrocellulose nylon membrane fixed is put into hybridization bag
In, it is added after appropriate hybridization buffer and carries out prehybridization 15min under the conditions of 50 DEG C；100 DEG C of the DIG probes that embodiment 5 is synthesized
It is added in hybridization buffer after being denaturalized 5min, 50 DEG C of 1.5~2h of hybridization；Film is placed in 2 enough × SSC (containing 0.1%SDS) solution
In, oscillation rinsing 2 times, each 10min；It is transferred in the solution of 0.5 enough × SSC (containing 0.1%SDS), it is light under the conditions of 50 DEG C
Micro oscillation rinses 2 times, each 10min；Buffer2 is added, jog closes 20min.
6.2 immune chromogenic reactions
It is 1 ﹕ 7500, room temperature that the-DIG antibody (Roche companies) that AP (alkaline phosphatase) is marked, which is added, to reach working concentration
It is incubated 30min；Film is washed with Buffer1 2 times, each 10min；It is added in 10mL chromogenic substrate buffer solutions；Dark place colour developing about 10~
Film is rinsed 5min, color development stopping reaction by 30min with 50mLbuffer4.
The specific detection of embodiment 7.NASH methods
The viral nucleic acids such as extracted WYSV, WDV, BYDV-GAV, BYDV-PAV, WYDV-GPV and BYSMV are respectively taken 1
μ L points are on same nylon membrane, using healthy wheat RNA as negative control, with the WYSV specificity DIG nucleic acid probes of preparation
Carry out nucleic acid spot hybridization.As a result it shows：The probe only reacts (Fig. 8) with the RNA of WYSV samples generations.Show that this probe has
Stronger specificity can be used for differentiating and distinguish other viruses on WYSV and wheat similar with its symptom.
The sensitivity technique of embodiment 8.NASH methods
2 parts of WYSV sample RNA are extracted, number is W1 and W2.Nucleic acid concentration is measured with Nanodrop microdetermination instrument, respectively
For 1121.33ng/ μ L and 1530.08ng/ μ L.1 μ L RNA are taken respectively, then press 10-1~10-6Serial ratio ddH2O dilutes
RNA, then by dilution sequence point in carrying out dot hybridization on same nylon membrane.Hybridization colour developing result (Fig. 9) shows 10-2Dilution
Multiple can obtain more clearly RNA traces, and 10-3Extension rate remains to detect RNA, the average sensitivity of 2 repetition experiments
More than 1ng/ μ L.
Embodiment 9. is applied to the detection of the doubtful WYSV samples in field
Wheat growing season in 2017 collects disease in 6 provinces such as China Shaanxi, Shanxi, Shandong, Yunnan, Guizhou and Hubei
Doubtful 100 parts of the standard specimen for WYSV viruses of shape.Every part of standard specimen weighs 0.1g blades, using TRIzol (Invitrogen, USA) method
The total serum IgE for extracting wheat samples to be detected respectively according to embodiment 3 is dissolved in the ddH of DEPC2In O solution, -70 DEG C temporary standby
With.Field sample to be tested is detected using the NASH technologies established in embodiment 6,100 parts of doubtful standard specimens of WYSV are detected.
Take 1 μ L points on nylon membrane the standard specimen RNA extracted, the information and testing result of sample are shown in Table 2 and Figure 10.NASH result tables
Bright WYSV in 2017 occurs in Hancheng Region, Shaanxi and Hubei Wuhan Area spot film.
2 NASH methods of table detect the doubtful WYSV viral samples in field
Sequence table
<110>Plant Protection institute, Chinese Academy of Agricultral Sciences
<120>Wheat Yellow strip virus and its NASH detection methods, detection kit
<130> PP18012-ZWB
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 1
ggtgctcagt gtcgctttct 20
<210> 2
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 2
acctgctgtt ccattcttgt c 21
<210> 3
<211> 14486
<212> DNA
<213>Unknown (Unknown)
<400> 3
acaccaccag acaacaactg cacataggat ccagtattta gaatgtttgt cggcagatga 60
gcaaatactg ggaactatgg acacgtcatt gtctgggtcc caaaagtacc tgggcccacg 120
caagcgtcgt cacattccta atcatggaaa aaacatcact cacaggcaag gggcatcatg 180
ttcctctatt tctcatttta aaaaccccaa caacaacttc actggcacca tggctcagaa 240
ccccaatgtt gctaactatg ctaatgctgc accactcccc aggtttgaag gtctaggcga 300
cagagagaac cttgcaccaa tcggcaatga agcagtcgag ataccgtacc agaaagaagc 360
ctatctcgcc tggatcaacg aaggaagagt attccaggtc aatcagctta ctgatgagca 420
aatgattcaa atgtgggaaa ccgtcaagac atccatgcaa ggcaacacat tcagcgagca 480
gcacatgcgc gacattgtcc aaatggcctg caacctaaag ggcgtggacc cagccacaaa 540
gcccctctac aggcaatacg agatgccaga aaacggacgc tgggcagacg ctccctctca 600
ggatcccatc ttttcaggtc aacaagtagc aggagttata gtaccacttc aagaagcaca 660
gcccctagtt gaggatgtat ccgggaaagc tagagcaatt ggtttcatat gcggatttct 720
gttgaggttc atagtaaaga ctgaggagca tctgaataat tccttggcaa acttgaagct 780
ccaattcagt aggatatatg gtgtgcagtc tgcaaccata aatcaatgga acccaactac 840
cacatgggcc tctaggatca agcttgcatt tgacacctat ctgaccctgc gagcaaccgt 900
tgccttgcat gtggctcttg ctgatggaaa cctcaatgca gataatgtga acttcggtct 960
atgcagaatg ttggtgtttc aacacctaga gctgagtgga ttacagctgt ataaaatgac 1020
aatgactctg atatctcacc tcaatttgat atctcctgca aagtttctat cttgggtgta 1080
tgaccctctg gctgaaaaac cgatcacaca gatctacacc atagcaacaa cacatgacac 1140
cagagacaga caagaccaga aacactggaa atatgcaaag ttagccagag gacagtattg 1200
gctagacacg accgtcaaaa ggaaccaatt ctttgcatat gtcctggccg atctggaggt 1260
aagatatggt ttgggtggca agaccgagta ttcaaatcct aagaggatga aagctttgga 1320
cggaacacct gtcgagacaa gaaacgatgc tgagtcggtg gcagcagcct tccaacagat 1380
gtacagagtt attgaagatg agaagagaag agaggccgga gccgccttcc gtctggccag 1440
aggcatgcct cctcctccca atcctcctcc agcagcagga gggcagggag gtggtgttgg 1500
agcccaagga cttggcaatg tccaagctcc cccaggtgca ggaggtcaag tgatacctcc 1560
cggaaatctt ccacctccac cagcagcccc gcctgcaggc gcagccggac aggcgccagg 1620
ccaacaggac cagcagatca accctcctgg acaggtgccc atggatctgg accaaagggc 1680
aagagatgct gctgctctgg gacttgttta acgccagaac tagagcacaa tgctaatatt 1740
tatgttacct caatgtgatg gttgtgtgcc tttaaaatgg gttttatgtt gtgatatatt 1800
tatgtctagt ccgatatacc tctatatttt aagaagggta atgtatcagt attaacaatg 1860
tggtatgatt tatgttgact aagcatttaa ataaacttat gtatacgtgt tgtacctggt 1920
attgcagtat cttgagaata tatttggtga tatttaaaaa ccccaacaca atatttgatc 1980
actcttgcta gatcattaat tgcaagacag catcaaagat cagacaaatc atccaggcac 2040
tgacagcaac gcaacaagta gattgtccaa actatgtttt agtatggctg atagtttgga 2100
cacagacagg ggcagaacta caaggtcagg atcaaagtta aaagctcaga caacacatag 2160
ggtggggaca tcctctggat cagttcgagg cggaaaacca tacgagaaag acttcaaagc 2220
cgtagaagca aggtttcaca actttgatcc aattagtgca agcataatcc gaggggatca 2280
ggaaggaacc aaagcagacc taataatgag tgactcctca gtaacgaaag ccacagcacc 2340
ggctgaagcc gcggcgcccc cacccccgcc gcagccggcc gcagtccccc tcacaccacc 2400
agcaagtgca accacccagg ggcagaagag accaggagat ccagaaactt tggaggaggg 2460
gaatgctgct aaaagagttc aacgggcaaa caaggtgaac aacttattaa cgacacaagg 2520
aataggcgag ccttccaaat cagctatatc ccaatatgtg gttgctaggc tgaatgcaaa 2580
taatgtggag catgatgatg ctatggtggc tgaatgtgca aatatggcag tttacgggtg 2640
gaaagaaggg aagaaatttg tgtctacgca aatattgaac caggccacca cggtgatacc 2700
agatctaata acctccatgg taacgaatgc aaacctcatc accaatgctg caaatgcctt 2760
gaacagcatc ccggacaaag tcgcaggggc tatacgtacc aacatagaac atgtcgcatt 2820
tcagactgga tcaaaggcca caaagagaga cacattagta aggacggcag agagcattta 2880
tcaaaatgca gctgctgaat cgaaggtgga ctttatcaac aatttcataa tatcagcagg 2940
gatcaatata caagaagtta aaagaaatgt tgcaaattat aggacaattg caaatgcgat 3000
aataaagaga aacactgtat tgaacataat caatgagaca acggagcatc aggctctgct 3060
tacacaggtc cagggcaaca aagatgatat caggaacatt gcacgaggac tctcagcttc 3120
atatgttatc tagaggtacc tacgaataat aaaagttgtc gctgtatgct cagcagacta 3180
ctggtgagat gcagtgtcaa ccatctatgg tcggttgttt tgtaatctgt cttaatataa 3240
ttgtatccat ctttgtgttt catatagcct accgggcact tgtctcattt gggtcagaca 3300
tagcatgccc ctaaatttaa tattgactaa aggggaggtt gttacacatg tattctaaat 3360
tcacatatgt atccttgtta tatttaaaaa ccccaacaga gatatacagc ttgcacagca 3420
ggcagccaaa cagcgaggag cgcccgaaag acagaaagac aaccagcatg gcaagcgaag 3480
caaagaattc tcagaagttt tccttcagga acactgaaga ggaggttgat ctatcaattt 3540
ccaagtttgc cttgttcaag ctgaagctca aacagtctcg tgttgttaca atgttcggca 3600
atgatgacaa ggttgaccct gcaaactgtt acataaatat gaccagtata aagatagtca 3660
ctagcagtgt acttccagag agtgacccga gatttatggt gtgggagatg tcatataaga 3720
cagatgaaga gaatcataag ttagggcagc tggcctggaa agcatcatac aatggatcat 3780
ttgtcatcaa atccacctat gcaatgatgg tagtaaatgg tgatctatac accccgtata 3840
ctgccagcat aactacatca gatggtagcg aaatcaaagg tgttaaggta aacgtgatac 3900
ttaactggac accatcagat gtaaggccat caaatgctaa gatgggaggg ttcatgagcg 3960
acatatattg caacaacccc tcaaatggga agacacagat acctcctatg gtggcatggt 4020
atatggggaa ggacgagcag agatattgca agataatcaa taagttggca acagaattga 4080
gcagcgagaa catttatccc atgatggagc tcatatcaag cccacaaact tctctgaacc 4140
caatactgaa taggctgatc tcaagttccc tcgagaaggg tgatagagac agaatcagcc 4200
agatgaccaa agctgtcgga gggggcatgt cactgaacaa ggcagacata tcatatctca 4260
aagggattat tgagaaaaaa tcatcttctg ctttagtgac atttcttatg agagccagca 4320
cagagctggg tgtagaagtg tatattgatg gttgaacaaa gggagagata tattggagca 4380
ttattatctt ccctgacaac ttatatggta atagtaacac ccgcataagc acccactata 4440
ctatcgtcca ccaaaagccc aaacatcaca atatccgcag tgtgttgtac tattattatt 4500
ataatattta aaaaccccaa ctatactgaa tcaacaatca cacatcacac aaacagagta 4560
ccatggccca cagcaagata cgtataacgg gagctgggct agatggggtt tcttatttgg 4620
agagaatacg gaaatctcta tctctcaacc cagccgatta tcatgatgac agtgccgttc 4680
gtatctctct gagcttctat atcaagatat ccttccatga tgtatcagat tatgacgtat 4740
ttatcagaga aggtgttaca accacagagt tgtttgatgc tctgaagaca agctggaaac 4800
ggaacccaga gaatgtcaga tacatagatg ggatgaccca cacagatgat gaaatagata 4860
ccacaatatt actatgtgag ctcgttacta tcttgaaaga tctatctcta cacagaagtg 4920
atgagagaac tcattatagt ttaatgagca catccttgac actgggtttc ggagatcaaa 4980
taacacagcc acatgacaat gctgtaatcc ccatagtcac aacaaaggtg attccaagat 5040
acatgcacac tgttatacaa tatgagtatc caagagtatc aggcggtata gcggcctccg 5100
tgtgtgcagg gatatgtatc aggtcacctc ctatagggaa gtgtccgccg ataatgaaaa 5160
cagtcaatgt ggaagtgcta gcattccatt atggactaga tgccggccaa ggaccgcagc 5220
tggaggagac aaatgccact ccaaaggaag aagaactgaa gaagattaat ggattcaaac 5280
gaatgactac actgaacaag tcatcacggg ttatcaagaa gccaagcaag gagggagttg 5340
gagcagcatt gagcagaatg ctctcttgga agtgatgtca tattcacaag tagacggttt 5400
ataatgttat cctggtatat taatgttatg tgtaatgctg tagttttcta aatataccaa 5460
ataaagttat aaagataata taagacttat atttaaaaac cccaaccact atcttaacat 5520
cacaagtcca tcgtgaaggt cagaagatca caaagcagtc ctcaatcagg gaaagggaaa 5580
atgaggacca caaccagaat gttgactata atcatctgca tgttgtttgg actatacatg 5640
atcatgttag gggagaacat gatactagca ttcggccctg tggacggtga tgatgactcg 5700
caggagaatc ggcccttcac actacatcct gctcacggag cgattccggt tttaccttcc 5760
agtcagccca gccagaaagt cagtcctgat gacaccaact cattaataag attgacgaca 5820
tcggaacagt tatggcacaa tcaccctcaa gatataggtc caaaagatgt cccagcagac 5880
atgtatccga tctattcctg tcctaatcta tcaaatgctt acttgttgcc gatatggtat 5940
ggaagttgcc ttgatgcatg ccagataaca acccccaaac atacagtgaa tgtgaagctc 6000
tggactatca acagctcagt aacagatgtt gacgggtatc agatagatgt gtattatgac 6060
acaaagttca gtcatgtcgg gccatttggt gggtgctcag tgtcgctttc tgagtcagtg 6120
acaaaagagc cgaagcagga ggatattatg atctggaaat caagactagt tagtaagccg 6180
gtgaacgatg tagaaagttg gatcatgtat gatgagccta gttgcaacta cttctctgat 6240
gagtactctt cagggttcag acttgtaatc acgagaacaa aattgaagct gatgatagac 6300
tcggttggaa atctttatat agcagatctg aggccagggt catatgatac atataaaacg 6360
ggatatgcaa tacatggttc tacagcctgg atatgggaca cagatgacag catgaaccat 6420
ggaatgtgtt acttcaagca gacagatgat acctattgcg attatgataa taacactaaa 6480
tatatgttct gcaagatgtc aggggtatca ttcgacacaa ctgtgcagca aagaatcacc 6540
tcaagttgtg ctggggattt gaacatatct acagatggag taatatatca aataggggac 6600
agtggtgata cggcctcgac acagcagaga ctatcagaca tattgcacca gaatgtcgag 6660
ctaggaatgc aatctctggt atccttgatc aatgatgtct ttataaacat tgaatcttca 6720
tactgtaccg gtgtttgcga tataatggag gtgattgtga gcaactaccc tacagcaaca 6780
actgttttag aaacaccgat aggaccatgg ctgcccataa catcagatgg acacaccatc 6840
atgactcctt gcatggctga tgttaattgg atcatacaga cacctatcgt ttattgcttc 6900
agcaaggaga tgataaaagt catcaacaaa gacacaagaa aggaagcctg gtggaggata 6960
gtgaactcgt acatcatatt gaatgagaca tgttcagata caaattcaac agccttggag 7020
atccttagag atcggatgtc taagaggaga gacattgtct acagcttttg gagaggggac 7080
ttgatagtgt cttatccata taacaaatca aggtggataa catataaaga cgagaagatc 7140
cagagaagct ccaaatggtt tgataaattg gtggacttga aatacaagca tcctataaca 7200
ctggataata tcacgagtca gcttgtgaac catacagctg acctatatga atggcatatg 7260
ggggacaaga atggaacagc aggtcagaca acattctcag atctcctggg aagagttgaa 7320
aaagcaggga caaacgtgat caagggatgt gtgaaaatga cggggaacct attgatatgg 7380
ataacgtccc atattgagat gattggtgat atgctaatta taatagtgtg tttgatcgga 7440
ggctattatg ttcttattat cccttatgga tttctgagga gagggagagg cccgggcact 7500
gtcacagaag ttcagcagtc gaacagtcag ttcagatctc ccttaatacc taggacatat 7560
ctgtgagaag gggacactaa aaagtatatg gactagccta agtggtgcat aatgcatctg 7620
actatcatat ttatagtaaa tgttcctgag ccggataagg gatacatttt tatatcttgt 7680
gcattatcca catatacttt actattatta atagattcat tatagtaaat tgtatttaca 7740
aaaaccccaa cgaagaacat ggacaagagc ctagacaaca agcaaggaga ccttctcaag 7800
atgtcaaaac ccaccgcaga cacaggcagc ccagacacgc cacaccccga gccagcacac 7860
accccagagg gggacacaac ccaaaaagaa gacacgaagg gaaacacagg caaggaagaa 7920
gcagcccaga gccccgaagc ggagcaagag gatgacaccc agccagaatg gtgctgcgag 7980
atagaagagc tcgatgttga gtcagactgg gaattttgct atcgagagga agatttcgac 8040
tgttactttg ataatgtatg ggtatatgag ttaattgatg aatgcaatgg atggagggaa 8100
taaatagtgc atgaacgatc catcagtgat atccacgtcg caacttaaag gaatctggct 8160
ccaggggagt taatataaag tttctttaaa taatcagaat caatagattc aatgagtgtt 8220
gtgtgatgtt gggttgctgg atatcaagac tgattatgta tcattatgtt gtgtgtatat 8280
ttaaaaaccc caacgaatga ttcatcattc acagagtaga gatccaaggt atggatgttg 8340
aagaaccaac atattgggca cacgatgaag acgatgacta ctggtgggat gcagatgagt 8400
acttgggtga agaagaagaa gatgaaatct atgaagatac agaggatgaa ttggtggagg 8460
ggggagactt tcatctaaaa tctgcgctaa gaggggaaga agacatgttg cagaatccta 8520
tttacaagaa agagcgggac agtatggttg aggagctagg gagtctaggc acattgatga 8580
accatataga tgttgtatcc atgctgaatt tgattggaac tagggttgca caggacaaac 8640
ggccgagtgg aggacatact ttcctacatg aaggggggtt caatataata cctttgcagg 8700
aaaatgtaaa gctgataaag gctgagctca tgtctgtgtt accagagatg gctgggatga 8760
atattgacca agtgttgtca gggtcatatc atctgttcat ggcagatcac ccgtatgtca 8820
cctgtacaat aagcactgtc ctgttcatac tctctgtgtt gaacaatgta aaaaacatca 8880
gacaaggtat cgaggttggc accctgatag gaaggattct atacaggaaa ggaaatcttg 8940
tatgtttaag cttagctgca gcaacattgt gttatctatc aactgatata ctggtttttg 9000
atgttgatgg ctcacgacat tacatgccga agacctattt cctcaacggg tgtgacaaag 9060
ttcaagaaag attcaatatc atgttgtatt catacatggc tgagtcattg ggagtaccgg 9120
gctcatgccc atctcagata gtgaaaagaa caatatcatg gggagacaat gtgttggcta 9180
acatggggaa tgaaggatac aatgtcattg gactatatga agctatactg gtggggatga 9240
tattagatag agatgatgaa gcattatctc ctacacctcg atcagagtcg tttctgtcaa 9300
atatattaac aggactgtca tcagaggaac aaggatacgc aaagcggtta atcaccacat 9360
tgcaagacat gagtccggct caattagctg atttacacgg tctatatagg atatggggtc 9420
atcctataat agatattgac ggcggggttc gaaagctaca gagagttacc agagaagaga 9480
aaggtgatat aaatgcaaag ccagagagca gagaaacagt gcgttcattc aggaggctct 9540
ttaccacaga atacttcaag aaacactcca tttatccccc aatggagata acatctaagt 9600
tcaatactta cttgggcaac tgcatccgaa caggaaaaga aattgatgaa aaacatataa 9660
attaccagtt ctctgattgg gactacattg aattgcagga aacattctcc attccatact 9720
catggaatgt gctgcatctt gcaaaggaca aagcaatatc tccaacaaga aatgagatat 9780
acaacatgct cctaagcaaa ggcaggatat tcaacgcaga gttgcggaga ggcgttctta 9840
aactgatgac aacaacactt atcccgttga gggatttttt gaagaaggtg gcatcagatg 9900
ggttagacat agatgattgt atcatcggtc tatttcctaa agaacgagag ttgaagatac 9960
ttgccaggtt ctttgccttg ctgtccttca atatgaggtt gtatttcacc tcaacagagg 10020
aactattagg aagtaaactt ctgaaatact ttccacagat aactatgagc tctaatttgt 10080
tggaaatgca ggaaaagatg gcttctatgt cgaaagaact aagaactcag aacaggtctg 10140
tgacatatgt gataaacatg gattttgtaa aatggaacca acaaatgaga gaatcaacat 10200
gccgaggggt cttcactgaa ctggataagt tatttggatt gaaaggcttg tatacgagat 10260
ctcatcaaat attcaaagat agtgtcttgt atatagcaga tggaacacgc agaatcttgc 10320
ctgacccaat atctggcgta atgatagatg acaatgcatg ctggacggac gacggagcag 10380
gcaaagaggg tatacggcag aaagcctgga ccataatgac cgtgtgtgac attgctgctg 10440
tggccaggca tcaccctggg aaattccacc ttgtgggagg aggagataat caagttctaa 10500
caataactta tcacacaaac caagtagata cgcagggggt cataactgag gcagggaaac 10560
aaaaaataaa aagcaaggta aagagattct tgacagatct agagaaacac ttttcagaaa 10620
gggggctccc attgaagact acagaaacat ggtgcagtac atctcttttc atgtataaca 10680
aatacatgta ttatcaaggt tctccacttc gttcaccact gaagcaggta tctaggcttt 10740
ttccgttctc gaacaacacc tctatgacct tacagtccat ggcacagtgt ttgggaacag 10800
gtctgagatc agttgcacag aaagaattat cacatatacc agcattaatg atgagaaata 10860
tctggggttc catcctaaca tggattgtca ctgtctgtca tcccatgttg ttaagcatct 10920
ctaaccaaga cagcttgaaa ggagacggta tcataactag ggggaaaaag gaaataaaaa 10980
tgaaagtgga atccgtggag tcggctccgc tagccttgaa gataatttat ctgccaggac 11040
attttggagg gcctggattg gttaatttgt tacaaatgac aatgagaggg ttcccagacc 11100
caattacaga aggaatttat ttcttgatgg ctatgaagaa aaatactgcg cgattgggat 11160
cagcatacac atctttattc cagagaatgg ccggagtatc attttcgagg agcaggaatt 11220
atgagcctct agttgaagat gtgtgttcac tgaacacaga cgctccaaga tccggaacga 11280
gtgaacagag ggaggtggcc aggaagatac tgttaaaatc aaaattagga gggaatgaaa 11340
atctaagaga cttattaaga ataatggaag gagacaatga gaagaatttc tacaaagccc 11400
tcactgcatc aagagttctg gatatcagag tgttacatga aatagcaagt gcaacactgt 11460
atgcaataac aaatacattc acctcaagag ttgatcgtac tgccactcta aaaagactca 11520
cattgagata ttccatgata aagagcctag cggagtcaga gaggaaattc ctgaggtacc 11580
tactggtgag ggatttgaag cagcatgaca tcatgtttga aaaatgcagt agagttacag 11640
cagatgagtg cagaacactg ggatggggta aacagatcat aggggtcacc gtggctacac 11700
ctttcgagta tttgagtgtg tctctgcgag agaaacatgt ctgtgacgga aataatatca 11760
ttgtaagact atcatcctcc gggaacaaaa aacaattggg tgaggtatta ggtccttgca 11820
agccttatct cgggacgtat accaaagaaa aattcaagat gacagaggtt gccgcagctt 11880
atggggatga agatgtatta accaaagcac tgagaattat gaagataata aattggaggt 11940
ttcctgaggg ctcaacaatg agcgagattc tgaaagcacc tttcagggct gttacagata 12000
tagatcctca aaggatgata caagagtcat ctgtgacaaa aggagattat gatcacagac 12060
gtaaaatgga ctcaagggtt catgggggta ttccaaattt tgtaatcaca ccactgtccc 12120
atatgagcat ctgcactagc acatggtaca agcatgctag aggggggaag aacgaaaata 12180
tccatttcca agcatgcatt atacagacta tgtataaaat cgtaatgctc cttatgaaca 12240
acggaaaatg tgatgaaatt atccacgctc atgaatcctg ctcaacatgc atatgtgaga 12300
taacagagcc ggttctggac ttagtaacac ccatgatgga tctgatattc cctcaattgc 12360
agaataactc actagtgtac ataccggaac aggccatatc atttgaccac tcaaggatga 12420
aagaagtgga ctatgcaaga agagtaggga tgctggactg ccacagggac acagattata 12480
cttggcttga cacgtacagt tctctatcat ggttgataat gtgcgatgtt ataggttgga 12540
caagaatgcc tcattcattc tactttatga tccagaatga tgtcgatcat tatctgttat 12600
cgatgtatct tatatgctta tggaaagtga tgaggaagga gtttgaattg ataaacacac 12660
agttggactg ggaacctatg atcaaggtgt actctacaca agaagggata acagtcttga 12720
gcaaccagat gggtttagta gtggggggct cattggaggg gggattggaa gtggacatga 12780
aagatatatg ctcagctttc aacacgctca ccatcacaca aatacctccg ttctcagtca 12840
accttgcatt gccaaagatc cacaaacagg ttgcagcatg gtggatgctt atggatgaca 12900
acacgctcct ctgcatagat tgtcataata acctgaacgg gtattgggat ggaacatcaa 12960
taaacaggag ggttgacaat gacctaagat gctacataca tgcagatgga gatgtgtccc 13020
cgagaataat aaatgcacac ataagcaatt tgtcgcaagg gctgatacgt ctgacaccgg 13080
ggggaacatc ccagagccat ccaataatta ttggaggaga taagctgaac aacctagaga 13140
cagtgccttc agttgaggat gaatggcctg atgtagagtc catcactttg ttgaaccaga 13200
taggggagac caatgatatt gatgggagat tgagattcat tattgagtca atgtcagtaa 13260
tcatgcctga tatagtagtt gtgagacctg atatgttgga tatcacactt gtaaaaacag 13320
ctagggagct gttaaaatgt gatgatggct caaaactcat aatatacata cttgtggaag 13380
aaccatcata cctagagagt gggacaatat atgagatgga gagggccttc ccaaatagtg 13440
aaacagtgga tgttcaattt gattacagga aggcaccggg aggactgcac aagctgtgga 13500
tcaatccaag ggaggaatct atacaaaggg ttgaagtggg agattggata tgcatatctt 13560
tcacggattt gatcaaacat catgacagta tcctggcaga tacagtattg acaatgaaag 13620
aaaggaggtt aggtagacaa ttcttggtga acaagggagt tggaagcttc aaaccgggag 13680
atttgggaaa gtatatattc tcatctgaat tgaatatgag ttacaagcag tcccgtcagg 13740
tggattatga gatcaacaag gtgatgatcc agaatcccac attggggggt aaatttatga 13800
tattgaagag aagatgttcc tgggcattgt tttcatcaag gtacgagatg ctaagagcag 13860
ttgaaagaat aaaaaagaac atgacagttg agggaaggtc aacaaaaacc aaggaatgga 13920
gcaagacaca tcaagatgat gttctgatac tgattatatc tatgctgatg agggctgatg 13980
aaaaagatga aagtgagata ttcacattgg ggtatgtaaa ctgtgatgtt gagaacttgg 14040
tcatgtaccc gaagtttagc agcacacagg tagtatctct gagatatttg gactactttt 14100
ggtttttcaa gagaatgtat gagtatcaag agaaaacatt tgctattcca tatactgaga 14160
tcagaacatc tctgaacatc ggctcgccca agagatcagg atacaacacc atcaacagct 14220
ccgaatgatc ggcatgtcaa accaccagag aggcggcagc cgcccggagg caagcgacga 14280
gccgcagacc ccccaaccaa ccacaactgc ccccttttat aaaaacccca atctctctct 14340
gtcaagatag agggccctta agggaagagg ccaagagagg aaagggatgc gccctgcccg 14400
tgtggctgtg gttttcttgt tgggtcttgg gataatgacc ccatatccat acaagaaaca 14460
agtttcggtt ttggatatgg tggtgt 14486
Claims (10)
1. Wheat Yellow strip virus (Wheat yellow striate virus, WYSV), the nucleotide of the coding viral gene
Full length sequence is as shown in SEQ ID NO.3.
2. one kind is for Wheat Yellow strip virus (Wheat yellow striate virus, WYSV) described in claim 1
Nucleic acid spot hybridization detection method includes following a pair of of specific primer pair in the PCR system reaction solution for preparing probe,
Its nucleotides sequence is classified as：
SEQ ID NO.1：5’ggtgctcagtgtcgctttct-3’；
SEQ ID NO.2：5’acctgctgttccattcttgtc-3’.
3. detection method according to claim 2, the probe is by marker of digoxin by made from PCR methods
DNA probe.
4. detection method according to claim 3,25 μ L of total volume in the PCR system, wherein 0.5 μ L recombinant plasmids, 2
WYSV specificity in the DIG-dNTPs of a concentration of 2.5mM of μ L, 0.2 μ L rTaq and a concentration of 10 μm of ol/L claims 2
Each 0.5 μ L of primer pair, the recombinant plasmid are the pMD-18T recombinant vectors of the target fragment of portion gene containing WYSV.
5. detection method according to claim 4, the recombinant vector is by the WYSV specificity in claim 2
The PCR product that primer pair is obtained with reverse transcription PCR amplification, is connected to after purification on pMD-18T carriers, conversion to 5 α large intestines of DH
In bacillus competence.
6. detection method according to claim 4, the pcr amplification reaction condition are：94 DEG C of 3min, 94 DEG C of 30s, 56 DEG C
45s and 72 DEG C of 90s is recycled for 35 totally, 72 DEG C of 10min.
7. any the methods of claim 1-6 are detecting the application in susceptible crop plant body in WYSV viruses.
8. application according to claim 7, the crops are wheat, barley or oat.
9. a kind of kit for detecting WYSV viruses, the kit include：PCR system reaction solution, NASH reaction reagents,
Nitrocellulose filter and hybridization bag；The NASH reaction reagents are composed of the following components：
Contain the WYSV specific primers pair described in claim 2 in the PCR system reaction solution.
Further include WYSV positive control samples and negative control in the kit 10. kit according to claim 9
Sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109678952A (en) * | 2018-11-29 | 2019-04-26 | 中国农业科学院植物保护研究所 | A kind of polyclonal antibody based on Wheat Yellow strip virus G-protein, preparation method and applications |
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| CN109678952A (en) * | 2018-11-29 | 2019-04-26 | 中国农业科学院植物保护研究所 | A kind of polyclonal antibody based on Wheat Yellow strip virus G-protein, preparation method and applications |
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