CN104195269A - Method for detecting tomato spotted wilf virus - Google Patents
Method for detecting tomato spotted wilf virus Download PDFInfo
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Abstract
The invention discloses a method for detecting a tomato spotted wilf virus according to a pyrosequencing technology. The method comprises the following steps: firstly, designing a specific primer and a pyrosequencing primer according to the gene N of the tomato spotted wilf virus; secondly, extracting ribonucleic acid of a sample to be detected, and performing polymerase chain reaction (PCR) amplification by the specific primer of the gene N; thirdly, detecting an amplification product by agarose gel electrophoresis, preparing a pyrosequencing single-stranded template if the length of a primer amplification fragment is 143 bp, producing pyrosequencing reaction, and finally judging whether the sample contains the tomato spotted wilf virus or not according to a PCR amplification result and a pyrosequencing result. The method is suitable for quickly detecting and confirming the tomato spotted wilf virus, and can be widely applied to epidemic situation monitoring in agricultural production and environment and confirmation of the tomato spotted wilf virus in import and export trade; the operation is very easy and convenient, and the required sample amount is small.
Description
Technical field
The invention belongs to the detection technique field of pathogenic, be specifically related to a kind of method that detects tomato spotted wilf virus.
Background technology
In recent years, tomato spotted wilf virus (Tomato spotted wilt virus, TSWV) has been listed in one of ten maximum kind of plant viruses of world's harm with its host range and tremendous economic loss of causing widely.TSWV is most important a kind of virus during TosPovirus belongs to, be distributed widely in temperate zone, subtropics and torrid areas all over the world, infect the single, double cotyledon plant of 82 sections kind more than 900, danger to vegetables, flowers and plurality of cereals crop, as tomato, capsicum, potato, peanut, Ge Ba, pea, tobacco, chrysanthemum, Anemone cathayensis Kitag., youth-and-old-age, gloxinia, Garden Dahlia and Cyclamen persicum etc., are caused heavy losses to many cash crop and garden plant.It is reported, this disease once caused the loss of 80% peanut, 50%-90% Pueraria lobota fragrant plant dead, and at European Regions such as France and Spain, along with amboceptor Frankliniella occidentalis determine grow and diffusion, this disease can produce crushing harm to tomato, capsicum and Anemone cathayensis Kitag., and loss can reach 100%.
The natural propagation of tomato spotted wilf virus between host plant plant is mainly to propagate in persistence mode by thrips medium, and this virus copies voluntarily propagation in insect vector body, has improved viral propagation efficiency.At present, a plurality of regions such as tomato spotted wilf virus Yi China Sichuan, Guangzhou, Yunnan exist, along with Frankliniella occidentalis and tomato spotted wilf virus cross infection more and more frequent, the quick variability of tomato spotted wilf virus strain and Frankliniella occidentalis all can cause causing serious financial loss because two kinds of Harmful species interact.Only have the Rapid Confirmation method of setting up tomato spotted wilf virus, by timely diagnosis with get involved in advance and fast processing, could, effectively in the further diffusion of propagating containment virus on source, in the shortest time internal cutting off, propagate chain.
At present, detection and confirmation method for TSWV generally judge by serology, biological host, morphology test whether plant has plant virus, these methods are time-consuming, complex operation, can not meet the needs of disease control, microscope inspection analysis simultaneously depends on personnel's experience and specialty background knowledge more, and subjectivity is larger; When application round pcr detects confirmation, if primer specificity is not strong, may there is detecting false positive; Because tomato spotted wilf virus variability is stronger, strains is that base difference degree is larger, and the primer having been reported is easy to because Mutation causes occurring the problems such as false positive, therefore, needs conservative property is higher, reach SNP level detection primer or detection method.
Summary of the invention
The object of the present invention is to provide a kind of method that detects tomato spotted wilf virus, thereby overcome the shortcoming that prior art exists on confirmation TSWV, seek to provide a kind of TSWV tetra-sodium sequencing technologies confirmation method, to overcome the deficiency of existing detection technique, for the monitoring of agriculture production and ecotope provides a kind of quick, easy, efficient, practical TSWV confirmation detection method.
First the present invention provides a kind of primer sets for detection of tomato spotted wilf virus, and sequence information is as follows:
Sequencing-F:5 '-TAAGGCTTCCCTGGTGTCATAC-3 ' (SEQ ID NO:1), 5 ' carry out biotin labeling;
Sequencing-R：5′-CCATAATGCTGGGAGGTAGCT-3′(SEQ?ID?NO:2)，
Sequencing-sequencing primer: 5 '-GTTGATAGCTTTGAGATGA-3 ' (SEQ ID NO:3).
The present invention also provides by above-mentioned primer sets to detect in sample whether have tomato spotted wilf virus, and concrete grammar is as follows:
1) the testing sample RNA of extraction is carried out to RT-PCR amplification, after reverse transcription, in amplification solution, add 2 * PCR buffer, 12.5 μ L, each 0.5 μ L of 10pmol/ μ L upstream primer and downstream primer, DEPC water is supplied volume to 25 μ L, and PCR cycling condition is: after 94 ℃ of denaturation 5min, enter 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 50 times; Then 72 ℃ are extended 10min again;
2) by pcr amplification product electrophoresis detection: get 2g agarose, heat in 100mL electrophoretic buffer, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, and glue adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed respectively to point sample with appropriate sample loading buffer; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates to gel middle part; Under Ultraviolet Detector, observe electrophoresis result record.
3) prepare tetra-sodium order-checking single-stranded template: use 50 μ l to be marked with the PCR product of vitamin H and the coated magnetic bead of 200 μ g Streptavidins, at 25 ℃ of room temperatures, hatch 20 minutes, with vacuum prep tool, the PCR product after being combined with magnetic bead is picked up, then in 70% ethanol, clean 5s; Denatureation buffer washes 5s; Finally move on in washing buffer and clean 10s; Then vaccum prep tool is put into the plate that contains sequencing primer, shake, discharge magnetic bead.Sample is put into 80 ℃ of baking ovens 2 minutes, then cool to room temperature;
Tetra-sodium sequencing reaction: sequencing reaction at room temperature detects on tetra-sodium sequenator, application of sample is used application of sample pressure and the time of 600 millibars/8 milliseconds, every wheel 65 seconds reaction times, primer strand extended along with adding of different dNTP, combination along with nucleic acid, ccd video camera detects the optical signal sending, and reads DNA sequence dna.
Of the present inventionly about judging that according to pcr amplification result and tetra-sodium sequencing result whether to contain TSWV:PCR reaction in sample positive, carry out tetra-sodium sequencing analysis, order-checking fragment and N gene target fragment are identical, are defined as TSWV; PCR reaction is positive, and it is non-TSWV that order-checking fragment and target fragment have an above base different decision; PCR reacts the negative non-TSWV that is judged to be.
The present invention is applicable to TSWV to carry out rapid detection confirmation, can be widely used in producing and environment in disease monitor, the confirmation of this virus in foreign trade.
Compared with prior art, beneficial effect of the present invention comprises:
The first, its application tetra-sodium sequencing technologies can carry out short nucleic acid sequences analysis quickly and accurately, is convenient to build normalizing operation flow process;
The second, there is high-throughput, feature cheaply, PCR product can be directly used in order-checking, does not need to carry out the secondary treatments such as product purification, operates very easyly, and required sample size is little.
The 3rd, sensitivity and tolerance range are higher, under the sensitivity identical with RT-PCR, can directly read nucleotide sequence, and its accuracy of detection can reach the level of single base, realize SNP (single nucleotide polymorphism) site is detected and identification.
The 4th, PCR and product are checked order to the two merges, change traditional harmful organism Molecular Detection and be sent to the pattern of biotech firm's order-checking, testing process was foreshortened to 2～4 hours by 4～7 days.
Accompanying drawing explanation
Fig. 1: be the amplification collection of illustrative plates of the present invention to N gene in diseased plant sample, M:DL2000 wherein, 1,2: diseased plant sample, 3,4: healthy plant sample;
Fig. 2: for the present invention is to N gene tetra-sodium sequencing result in diseased plant sample.
Embodiment
The present invention is hybridized by sequencing primer and polymerase chain reaction (PCR) amplification single stranded deoxyribonucleic acid template, hatch with deoxyribonucleic acid polymerase, adenosine triphyosphate (ATP) sulfurylase, luciferase, apyrase and substrate (APS), fluorescein, add one by one four kinds of triphosphoric acid base deoxynucleotides (dNTPs): (triphosphoric acid adenyl-deoxyribonucleotide dATP; Triphosphoric acid thymidylic acid dTTP; Triphosphoric acid deoxycytidylic acid dCTP; Triphosphoric acid guanine deoxyribonucleoside acid dGTP), as matched with template, the end of this triphosphoric acid base deoxynucleotide and primer forms covalent linkage, discharges tetra-sodium group (PPi); Adenosine triphyosphate (ATP) sulfurylase catalysis tetra-sodium in the situation that substrate (APS) exists forms adenosine triphyosphate, adenosine triphyosphate drives the fluorescein of luciferase mediation to transform to oxyluciferin, and oxyluciferin sends the visible light signal being directly proportional to adenosine triphyosphate amount; Optical signals charge coupled device (CCD) is collected and is converted into peak by software; The peak height of each optical signal with react in the Nucleotide number that mixes be directly proportional, adenosine triphyosphate and uncorporated triphosphoric acid base deoxynucleotide are degraded by apyrase, cancellation optical signal, and regeneration reaction system, utilize optical signal to transform the peak figure obtaining, the tomato spotted wilf virus in sample is detected accurately.
Below by specific embodiment, also further set forth by reference to the accompanying drawings the present invention.
Embodiment 1: detect in the sample of field whether contain TSWV
1, design specific primer sequence
By Internet, log in the state-run biology information technology of U.S. center (NCBI), the N gene nucleotide series of the TSWV that query and search has been announced, do not comprise not clear base components series, to the diseased plant of nearer same time of homology with place, with the Editseq in DNAStar 7.1 software packages and MegAlign software, the N gene nucleotide series of selected sample is edited, comparison one by one, with Clustal W Method (software) default parameters, selected N nucleotide sequence is carried out to homology comparison, find and characterize this viral specific nucleotide sequence (target detect sequence).
The design of pcr amplification primer and sequencing primer design all can be undertaken by Assay Design SW software, use one group of higher primer of score to test, according to the homology analysis result of DNASTAR, choose sequence area more conservative in sample gene, design of amplification primers, so that amplify the single band of specificity, for follow-up order-checking lays the foundation.For the specific nucleotide sequence (target sequence) comprising in each amplification region designs sequencing primer, N gene amplification fragment length is 143bp simultaneously.
TSWV PCR and sequencing primer are:
Sequencing-F:5 '-TAAGGCTTCCCTGGTGTCATAC-3 ', 5 ' carry out biotin labeling
Sequencing-R：5′-CCATAATGCTGGGAGGTAGCT-3′，
Sequencing sequencing primer: 5 '-GTTGATAGCTTTGAGATGA-3 '.
2, extract the RNA of tomato sample to be measured
Utilize the diseased plant of field morbidity, with phenol-chloroform method extracting, obtain; Also available business-like RNA extracts test kit extraction.
3, with TSWV N gene primer, carry out RT-PCR amplification
In amplification solution, add 2 * RT-PCR buffer (10 times of polymerase chain Mix reaction solutions), 12.5 μ L, each 0.5 μ L of 10pmol/ μ L upstream primer and downstream primer, the testing sample RNA of extraction is added in reaction system, DEPC water is supplied volume to 25 μ L, and PCR cycling condition is: 48 ℃ of 30min, after 94 ℃ of denaturation 5min, enter 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 50 times; Then 72 ℃ are extended 10min. again
4, carry out agarose gel electrophoresis
PCR product is carried out to agarose gel electrophoresis, get 2g agarose, in 100mL electrophoretic buffer, heat, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, glue.In electrophoresis chamber, add electrophoretic buffer, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed respectively to point sample with appropriate sample loading buffer; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates to gel middle part; Under Ultraviolet Detector, observe electrophoresis result, field to be checked sample can amplify specific band, and amplified band is 143bp, and electrophoresis result is shown in Fig. 1.
5, prepare tetra-sodium order-checking single-stranded template
Use 50 μ l to be marked with the PCR product of vitamin H and the coated magnetic bead of 200 μ g Streptavidins, at 25 ℃ of room temperatures, hatch 20 minutes, with vacuum prep tool, the PCR product after being combined with magnetic bead is picked up, then in 70% ethanol, clean 5s; Denatureation buffer washes 5s; Finally move on in washing buffer and clean 10s; Vaccum prep tool puts into the plate that contains N gene sequencing primer, shakes, and discharges magnetic bead.Sample is put into 80 ℃ of baking ovens 2 minutes, then cool to room temperature.
6, tetra-sodium order-checking
On tetra-sodium sequenator (PYROMARK ID), react, under room temperature condition, application of sample is used application of sample pressure and the time of 600 millibars/8 milliseconds, every wheel 65 seconds reaction times, and primer strand extended along with adding of different dNTP; Along with the combination of nucleic acid, ccd video camera detects the optical signal sending, and N gene tetra-sodium sequencing result is shown in Fig. 2, and the sequence reading is TCAGTGTTGT CTTGGCTATA T.
7, result is judged
The N gene amplification fragment length of field sample is 143bp, consistent with expection; Through tetra-sodium order-checking, the N sequence of mensuration is consistent with TSWV target sequence, so judges and in sample, contain TSWV.
Embodiment 2: detect in import tomato seeds whether contain TSWV
1, design specific primer sequence
With embodiment 1.
2, extract the RNA of testing sample:
With embodiment 1.
3, with TSWV N primer, carry out RT-PCR amplification
With embodiment 1.
4, carry out agarose gel electrophoresis:
PCR product is carried out to agarose gel electrophoresis, get 2g agarose, in 100mL electrophoretic buffer, heat, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, glue, in electrophoresis chamber, add electrophoretic buffer, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed respectively to point sample with appropriate sample loading buffer; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates to gel middle part; Under Ultraviolet Detector, observe electrophoresis result.
Therefore sample P CR to be checked does not amplify specific band, judges in the sample of import tomato seeds and does not contain TSWV.
Embodiment 3: detect in different virus sample whether contain TSWV gene
By tetra-sodium order-checking confirmation technology of the present invention, press detecting step to separation the nepovirus (TRSV) from import Japan sweet Stevia, be located away from the tomato black ring virus (TBRV) of Italian cucumber seeds, be located away from the cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) of Chilean watermelon seed, the tomato yellow leaf curl virus (ToYCLV) and the Tospovirus that are located away from tomato planting district, Qingdao are viral---and 8 kinds of strains close with TSWV genetic background such as garden balsam necrotic spot virus (INSV) and flag flower macula lutea virus (IYSV) detect, the distinctive amplified fragments of TSWV-N gene and corresponding sequence do not detected.Thereby proof there will not be false positive results when the virus of non-TSWV is detected.
Embodiment 4: the TSWV viral nucleic acid standard substance that detect different concns
1, design specific primer sequence
With embodiment 1.
2, the preparation of RNA standard substance:
According to conventional molecular cloning working method, first after PCR product electrophoresis, cut the gel that contains object fragment, with the gel-purified test kit of Tiangen company, purify, spend the night and be connected with 4 ℃, pGEM-T carrier, transformed competence colibacillus cell, converted product is coated with dull and stereotyped cultivation, the order-checking of the positive Hou Song Hua Da of picking hickie PCR Product Identification genome company, according to sequencing result, the positive colony of screening is inoculated into the LB substratum incubated overnight containing penbritin, after preparation plasmid DNA purifying, after Eco I enzyme is cut, utilize T7 promotor in-vitro transcription to prepare RNA template, after ethanol precipitation, in ultraviolet foranalysis of nucleic acids instrument, measure OD
260value, quantitative assay concentration is 5 * 10
6copy/μ L.
3, with TSWV N primer, carry out RT-PCR amplification
Standard substance are carried out to serial dilution, and obtaining respectively concentration is 5 * 10
6, 5 * 10
5, 5 * 10
4, 5 * 10
3, 5 * 10
2, 5 * 10
1, 25 copy/μ L, 20 copy/μ L, 10 copy/μ L, 5 copy/μ L standard substance, the standard substance of different concns gradient of take are template, each concentration is established repetition, carries out RT-PCR and tetra-sodium sequencing reaction, reaction system and response procedures are with embodiment 1.
4, carry out agarose gel electrophoresis:
PCR product is carried out to agarose gel electrophoresis, get 2g agarose, in 100mL electrophoretic buffer, heat, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, glue, in electrophoresis chamber, add electrophoretic buffer, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed respectively to point sample with appropriate sample loading buffer; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates to gel middle part; Under Ultraviolet Detector, observe electrophoresis result.
When the density loss of nucleic acid standards is during to 10 copies/μ L, the distinctive amplified fragments of TSWV cannot be detected, tetra-sodium order-checking also cannot be read corresponding sequence.When thereby proof the method detects the virus of TSWV, sensitivity can reach 20 copies/μ L.This show the sensitivity of tetra-sodium sequencing technologies and tolerance range higher, in the situation that there is the sensitivity identical with RT-PCR, can read nucleic acid sequence, its tolerance range is higher, can reach the level of single base.This invention can realize detection and the identification to viral SNP (single nucleotide polymorphism) site.
Claims (5)
1. for detection of a primer sets for tomato spotted wilf virus, it is characterized in that, in described primer sets, the sequence information of primer is as follows:
Sequencing-F1：5′-TAAGGCTTCCCTGGTGTCATAC-3′，
Sequencing-R1：5′-CCATAATGCTGGGAGGTAGCT-3′，
Sequencing-sequencing primer: 5 '-GTTGATAGCTTTGAGATGA-3 '.
2. primer sets as claimed in claim 1, is characterized in that 5 of described Sequencing-F1 ' carries out biotin labeling.
3. the application of primer sets claimed in claim 1 in the goods of preparation detection tomato spotted wilf virus.
4. for detection of goods for tomato spotted wilf virus, it is characterized in that, the described test kit that is prepared as, includes primer sets claimed in claim 1.
5. application rights requires the primer sets described in 1 to detect a method of surveying tomato spotted wilf virus, it is characterized in that, described method comprises following step:
1) the testing sample RNA of extraction is carried out to RT-PCR amplification, after reverse transcription, in amplification solution, add 2 * PCR buffer, 12.5 μ L, each 0.5 μ L of 10pmol/ μ L upstream primer and downstream primer, DEPC water is supplied volume to 25 μ L, and PCR cycling condition is: after 94 ℃ of denaturation 5min, enter 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate altogether 50 times; Then 72 ℃ are extended 10min again;
2) by pcr amplification product electrophoresis detection: get 2g agarose, heat in 100mL electrophoretic buffer, fully dissolve, adding ethidium bromide stock solution to final concentration is 0.5 μ g/mL, and glue adds electrophoretic buffer in electrophoresis chamber, makes liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed respectively to point sample with appropriate sample loading buffer; 9V/cm constant voltage electrophoresis, until bromophenol blue indicator migrates to gel middle part; Under Ultraviolet Detector, observe electrophoresis result record;
3) prepare tetra-sodium order-checking single-stranded template: use 50 μ l to be marked with the PCR product of vitamin H and the coated magnetic bead of 200 μ g Streptavidins, at 25 ℃ of room temperatures, hatch 20 minutes, with vacuum prep tool, the PCR product after being combined with magnetic bead is picked up, then in 70% ethanol, clean 5s; Denatureation buffer washes 5s; Finally move on in washing buffer and clean 10s; Then vaccum prep tool is put into the plate that contains sequencing primer, shake, discharge magnetic bead.Sample is put into 80 ℃ of baking ovens 2 minutes, then cool to room temperature;
Wherein tetra-sodium sequencing reaction is specific as follows: sequencing reaction at room temperature detects on tetra-sodium sequenator, application of sample is used application of sample pressure and the time of 600 millibars/8 milliseconds, every wheel 65 seconds reaction times, primer strand extended along with adding of different dNTP, combination along with nucleic acid, ccd video camera detects the optical signal sending, and reads DNA sequence dna.
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