CN104120193A - Primer and method for carrying out specific detection and absolute quantification on tomato spotted wilt virus - Google Patents
Primer and method for carrying out specific detection and absolute quantification on tomato spotted wilt virus Download PDFInfo
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Abstract
The invention relates to a primer and a method for carrying out specific detection and absolute quantification on a tomato spotted wilt virus and belongs to the field of plant virus detection. According to the primer and method disclosed by the invention, the tomato spotted wilt virus is taken as an object of study, a more rapid, simple, accurate and sensitive method for quantitatively detecting a plant virus is established, the method comprises the following steps of (1) establishing a standard curve according to the standard plasmid; (2) obtaining the cDNA of a sample to be detected; synthesizing cDNA by virtue of reverse transcription; and carrying out fluorescence quantitative PCR by virtue of using cDNA as a template and TSWV-113F / TSWV-113R as a primer to obtain the Ct value of the sample to be detected; and (3) calculating the copy number of the tomato spotted wilt virus in the sample to be detected by virtue of the standard curve. By virtue of the method, tomato spotted wilt virus within the sample can be accurately quantified in 4-5 hours, the time is saved and the experimental steps are simplified, the method is contributed to taking corresponding remedial measures before the onset of plant diseases to prevent huge economic losses brought by the widespread of the virus among crops.
Description
Technical field
The invention belongs to plant virus detection field, relate to particularly a kind of for tomato spotted wilf virus being carried out to primer and the method for specific detection and absolute quantitation.
Background technology
Tomato spotted wilf virus (Tomato spotted wilt virus, TSWV) be one of representative member of bunyaviridae (Bunyaviridae) Tospovirus (Tospovirus), this virus particle almost spherical, diameter is approximately 80-110nm, wherein adventitia is 20～25nm, nucleocapsid diameter is 60nm, there is coated triad single stranded RNA (the L RNA of film, M RNA, S RNA) genome, the 4 kinds of structural protein of encoding, the 5' of three fragments and 3' end have 8 conservative complementary bases, can form false ring texture.L RNA (～8.9kb) contains an open reading frame (open reading frame, ORF), coding viral RNA dependency polymerase protein (RdRp).M RNA (～4.8kb) and S RNA (～2.9kb) are ambisense RNA, M RNA viruses chain encoding Nonstructural Protein NSm wherein, complementary strand coding glycoprotein g1, the precursor protein of G2; S RNA viruses chain encoding Nonstructural Protein NSs, complementary strand coding nucleocapsid protein (nucleocapsid protein, N).At occurring in nature, the propagation of TSWV from plant to plant almost propagated by lasting modes of reproduction by Frankliniella occidentalis completely.
TSWV was found in Australia by Brittlebank first in 1915, was now extensively distributed in a plurality of countries and regions in the world, infected 900 various plants such as tobacco, peanut, capsicum, tomato, soybean, lettuce, chrysanthemum.It can cause serious financial loss to different flowers and vegetable crop, with its host range and tremendous economic loss of causing widely, has been listed in one of ten maximum kind of plant viruses of worldwide harm.At present, the main link of the prevention and control of viral diseases of plants is exactly the detection monitoring of plant virus.
From the initial detection that present numerous technology is applied to plant virus that observes directly, as serological technique, Electron Microscopy, Protocols in Molecular Biology etc.But along with improving constantly of testing requirement and standard, these routine techniquess are more and more difficult to meet the requirement of research, as detecting sample, traditional serology ELISA method conventionally needs 1～2 day, and normal PCR needs 7～8 hours consuming time.And real time fluorescent quantitative RT-QPCR is a kind of higher than conventional PCR sensitivity, more special quantitative tool, the method is only 4～5 hours from the interpretation of result time of extracting of sample, a kind of detection means fast of can yet be regarded as, and the quantitative data obtaining can highly repeat.This technology has been widely used in expression or the regulation and control of gene, and the relative and absolute copy number of quantitatively specific nucleic acid in different hosts or pathogenic agent, and this technology has demonstrated absolute advantage in the virus of plant tissue detects.
The detection method of fluorescent quantitation is divided into relative quantification and absolute quantitation, relative quantification is the amount of measuring respectively goal gene and reference gene, obtaining the relative quantity for the goal gene of reference gene again, finally carry out the comparison of sample room relative quantity again, is to goal gene a kind of method qualitatively.And absolute quantitation to be absolute magnitude (copy number) to unknown sample carry out method for measuring, can not only carry out goal gene qualitatively, main is can be quantitatively, more sensitive and accurate to viral detection.
Summary of the invention
The invention provides primer and the method for a kind of tomato spotted wilf virus detection and absolute quantitation, take tomato spotted wilf virus as research object, set up quicker, easy, accurate, sensitive plant virus quantitative detecting method.
The claimed technical scheme of the present invention is as follows:
A method of the plant sample to be measured of doubtful infection tomato spotted wilf virus being carried out to spotted wilt virus absolute quantitation, comprises the steps:
(1) prepare spotted wilt virus plasmid standard typical curve, step is as follows:
To determine the positive sample of plant that infects tomato spotted wilf virus, extract total RNA, reverse transcription becomes positive cDNA; Take positive cDNA as template, with tomato spotted wilf virus Auele Specific Primer TSWV-482F/TSWV-482R amplification tomato spotted wilf virus specific fragment; Reclaim this specific fragment and be cloned in conventional skeleton carrier and obtain plasmid standard;
Survey the concentration of plasmid standard, according to the copy number of plasmid (copies/uL)=6.02 * 10
23(copies/mol) * plasmid concentration (g/uL)/plasmid molecule quality (g/mol), draws the viral copy number of plasmid standard;
Gradient ground dilution plasmid standard, with fluorescent quantitation primer, TSWV-113F/TSWV-113R carries out quantitative fluorescent PCR to the plasmid standard of different concns, draws the Ct value of each concentration plasmid standard;
The logarithmic value of viral copy number corresponding to each concentration plasmid standard of take is X-coordinate, take Ct value as ordinate zou Criterion curve;
Described tomato spotted wilf virus Auele Specific Primer TSWV-482F/TSWV-482R, sequence is:
5’-TCTGGTAGCATTCAACTTCA-3’，
5’-CTTCTCTGGTGTCATACTTCT-3’；
The sequence of described fluorescent quantitation primer TSWV-113F/TSWV-113R is:
5’-CTTGCCATAATGCTGGGAGGTAG-3’/5’-TCCCGAGGTCTTTGTATTTTGC-3’；
(2) extract total RNA of described plant sample to be measured, cDNA is synthesized in reverse transcription; And take cDNA as template, TSWV-113F/TSWV-113R primer carry out quantitative fluorescent PCR, obtain the Ct value of plant sample to be measured; Described Ct is brought into the copy number that calculates tomato spotted wilf virus in plant sample to be measured in described typical curve.
The system of described quantitative fluorescent PCR is: template 1uL, 2 * SuperReal PreMix Plus10uL, each 0.5uL of 10umol/L fluorescent quantitation primer TSWV-113F/TSWV-113R, 50 * ROX Reference Dye5uL, RNase-free ddH
2o3uL, cumulative volume 20uL.
The reaction conditions of described quantitative fluorescent PCR is: 95 ℃ of denaturation 15min, and 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 72 ℃ are extended 32s, 40 circulations.
The concentration of described survey plasmid standard adopts Nano Drop2000c.
Described conventional skeleton carrier refers to pMD
tM18-T.
The application of above-mentioned either method in tomato spotted wilf virus absolute quantitation.
For detection of the specific PCR primer of tomato spotted wilf virus, its nucleotides sequence is classified as:
TSWV-482F?5’-TCTGGTAGCATTCAACTTCA-3’；
TSWV-482R?5’-CTTCTCTGGTGTCATACTTCT-3’。
The method of whether carrying tomato spotted wilf virus for detection of testing sample, comprises the steps:
(1) extract the RNA of testing sample, cDNA (quantitatively arriving 1ng/ul) is synthesized in reverse transcription;
(2) take the synthetic cDNA of reverse transcription carries out PCR reaction as template, obtains amplified production; The primer of PCR reaction is TSWV-482F/TSWV-482R:
5’-TCTGGTAGCATTCAACTTCA-3’/5’-CTTCTCTGGTGTCATACTTCT-3’；
(3) agarose gel electrophoresis detects amplified production, if the size of amplified production is 482bp, in testing sample, carries tomato spotted wilf virus; If the size of amplified production is not 482bp, in testing sample, do not carry tomato spotted wilf virus;
The system of described PCR reaction is: cDNA template 2uL, TSWV-482F0.5uL, TSWV-482R0.5uL, 2 * ES Tap MasterMix10uL, ddH
2o7uL, cumulative volume 20uL;
The condition of described PCR reaction is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ of extension 45s, 40 circulations, last 72 ℃ are extended 5min.
The present invention has set up a kind of for testing sample tomato spotted wilf virus absolute quantitation method, comprises the steps: that (1) is according to standard plasmid Criterion curve; (2) obtain testing sample cDNA; CDNA is synthesized in reverse transcription; And take cDNA as template, TSWV-113F/TSWV-113R primer carry out quantitative fluorescent PCR, obtain the Ct value of testing sample; (3) utilize typical curve to calculate the copy number of tomato spotted wilf virus in testing sample.Method accuracy is good, highly sensitive, high specificity, reproducible, can in 4-5 hour, to the tomato spotted wilf virus in sample, carry out accurate quantitative analysis, not only save the time, simplified experimental procedure, also helped before plant morbidity and take corresponding remedial measures, prevent the loss of this virus tremendous economic that wide-scale distribution is brought between crop.
In further embodiment of the present invention, also provide preferred reaction system and the condition of quantitative fluorescent PCR: primer TSWV-113F and TSWV-113R are with equimolar ratio configuration (10umol/L); PCR reaction conditions is preferred: 95 ℃ of denaturation 15min, and 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 72 ℃ are extended 32s, 40 circulations.Carry out with this understanding PCR, amplification efficiency is the highest, and specificity is best.In other embodiment of the present invention, also can adopt other suitable PCR reaction conditions to detect.
The Auele Specific Primer of design according to the present invention, the present invention also provides the PCR method of qualitative detection tomato spotted wilf virus.And provide PCR reaction system and the program that is applicable to this primer.Empirical tests, the method can be used for qualitative detection tomato spotted wilf virus, has good specificity, as shown in Figure 1.
Accompanying drawing explanation
Fig. 1 .TSWV object fragment PCR amplification and specific detection,
Wherein M is 50bp ladder, 1 positive sample (the thorn apple blade with TSWV of artificial preparation), 2-4 is respectively Vegetable & Flower Inst., Chinese Academy of Agriculture Science's Hot Pepper in Greenhouse blade, Jining ring grain Pepper Leaves, Jining shrinkage Pepper Leaves with TSWV, 5-7 is respectively with the water melon leaf of watermelon mosaic virus WMV, with the tobacco leaf of tobacco mosaic virus (TMV) TMV, with the melon cucumber leaves that moves back greenish-yellowization virus disease CCYV, and 8 is healthy Pepper Leaves;
Fig. 2. utilize the specificity of grads PCR checking fluorescent quantitation primer, wherein M represents Marker I, point sample hole 1-8 be quantitative fluorescent PCR product (annealing temperature: 54-62 ℃), the negative contrast in point sample hole 9;
Fig. 3. the amplified fluorescence graphic representation of plasmid standard;
The typical curve of Fig. 4 .TSWV fluorescent quantitative PCR detection method;
The amplified fluorescence graphic representation of Fig. 5 .TSWV fluorescent quantitative PCR detection method;
The solubility curve of Fig. 6 .TSWV fluorescent quantitative PCR detection method.
Embodiment
Virus strain: tomato spotted wilf virus TSWV-YN, is provided by Institute of Plant Protection, academy of agricultural sciences, Zhejiang Province and the Zhang Zhijun of institute of microbiology.
Also there is preservation in this laboratory, and applicant's statement in 20 years, can be provided for necessary proof test to the public from the applying date.
Vegetable material:
Thorn apple blade with TSWV: the method by frictional inoculation is kept at tomato spotted wilf virus TSWV-YN on thorn apple plant, detects and confirms morbidity through DAS-ELISA, standby.
The doubtful plant leaf with TSWV: take from Vegetable & Flower Inst., Chinese Academy of Agriculture Science's warmhouse booth, reveal the symptoms such as patch shape chlorisis, yellow, necrosis and calcination shape by judgement blade table and determine whether it is doubtful band TSWV.
Key instrument and reagent:
S-1000Thermal Cycler PCR instrument, Bio-Rad company;
Molecular Imager ChemiDoc XRS System.: U.S. Bio-Rad company;
Quantitative real time PCR Instrument 7500 types, American AB I company;
Refrigerated centrifuge 3K15, U.S. Sigma company;
TRIzol Reagent RNA extracts test kit, Invitrogen company;
The small-sized extraction test kit of plasmid, SuperReal PreMix Plus (SYBR Green), from TIANGEN Biotech (Beijing) Co., Ltd.;
DNA purifying reclaims test kit, purchased from Shanghai Jierui Biology Engineering Co., Ltd;
CDNA the first chain synthetic agent box, pMD
tM18-T Vector Cloning Kit, Takara PrimeScript
tMrT reagent Kit with gDNA Eraser (Perfect Real Time) test kit, precious biotechnology (Dalian) company limited.
The design of embodiment 1, primer is with synthetic
Step 1: design of primers
According to GENBANK database, report TSWV gene order, after TSWV sequence as much as possible (as JN601525.1) is downloaded, used DNAMAN software to carry out sequence alignment analysis; Virus-specific and the conservative region design primer (table 1) of finding different strains, primer is synthetic by Shanghai Sheng Gong company limited.
Object fragment primer is:
TSWV-482F：5’-TCTGGTAGCATTCAACTTCA-3’,
TSWV-482R：5’-CTTCTCTGGTGTCATACTTCT-3’。
Fluorescent quantitation primer is: TSWV-113F:CTTGCCATAATGCTGGGAGGTAG, TSWV-113R:TCCCGAGGTCTTTGTATTTTGC.
This experiment of table 1 the primer
Step 2: the specificity checking of Auele Specific Primer TSWV-482F/TSWV-482R:
Material:
The fresh thorn apple blade with TSWV,
With Vegetable & Flower Inst., Chinese Academy of Agriculture Science's Hot Pepper in Greenhouse blade of TSWV,
Jining ring grain Pepper Leaves,
Jining shrinkage Pepper Leaves,
With the water melon leaf of watermelon mosaic virus WMV,
With the tobacco leaf of tobacco mosaic virus (TMV) TMV,
With the melon cucumber leaves that moves back greenish-yellowization virus disease CCYV,
Acquired for materials from Vegetable & Flower Inst., Chinese Academy of Agriculture Science's plant pathology group laboratory above.
The amplification of the synthetic and object fragment of the extraction of the total RNA of plant, cDNA the first chain, chooses 1～3g vegetable material blade, with reference to CHMC's ultrafast type plant RNA extraction test kit specification sheets in ocean, extracts total RNA, uses TaKaRa reverse transcription test kit (PrimeScript
tMrT reagent Kit with gDNA Eraser) carry out cDNA synthetic, quantitatively to 1ug/ul, be stored in-80 ℃ standby.
Using the synthetic cDNA of reverse transcription as template, carry out the amplification of object fragment.Pcr amplification system is as follows: cDNA template 2ul, TSWV-482F0.5uL, TSWV-482R0.5uL, 2 * ES Tap MasterMix10uL, ddH
2o7uL, cumulative volume 20uL; Reaction conditions: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 56 ℃ of annealing 45s, 72 ℃ are extended 45s, 40 circulations, last 72 ℃ are extended 5min.
Get amplified production 10uL in tbe buffer liquid with 2% agarose gel electrophoresis, result is as Fig. 1, the single band that PCR product is 482bp, the material of the approximate symptom virus of other band does not detect band simultaneously, illustrate that the detection method of employing has good specificity.
The preparation of embodiment 2, the amplification of object fragment PCR and plasmid standard
(1) amplification of the synthetic and object fragment of the extraction of the total RNA of plant, cDNA the first chain
Thorn apple blade with TSWV is material, adopts the method for embodiment 2 steps 2 to prepare the cDNA with the thorn apple blade of TSWV, pcr amplification checking, positive cDNA sample is quantitatively to 1ug/ul, be stored in-80 ℃ standby.
(2) preparation of plasmid standard
The object fragment of the positive of step (1) amplification is reclaimed and purifying, be cloned into pMD
tMon 18-T carrier, place 30min on ice, add 50ul transT1 competent cell, then add 500uL not contain the LB substratum of ammonia benzyl, 37 ℃ of shaking culture, recovery 1h, is containing X-Gal, overnight incubation on the solid LB agar plate of IPTG and ammonia benzyl; Select white single bacterium colony spot, add 37 ℃ of LB liquid nutrient mediums, 200r/min shaking culture that 3ml contains ammonia benzyl to muddy; According to sky root plasmid extraction kit specification sheets, carry out plasmid extraction, plasmid is identified simultaneously, in-20 ℃, save backup.
The foundation of embodiment 3, TSWV real time fluorescence quantifying PCR method
(1) specificity of grads PCR checking fluorescent quantitation primer
This tests fluorescent quantitation primer used (113bp) is design in goal gene fragment (482bp) region.
With the TSWV thorn apple blade RNA that fluorescent quantitation primer pair extracts, carry out reverse transcription and RT-PCR amplification.Step is with embodiment 1 step 2.In PCR program, annealing temperature is made as 8 gradients: 65 ℃, 64.5 ℃, 63.5 ℃, 62 ℃, 60.2 ℃, 58.9 ℃, 57.8 ℃, 57.0 ℃.
Get amplified production 10uL in tbe buffer liquid with 2% agarose gel electrophoresis, result is as Fig. 2, the PCR product under 8 annealing temperatures is the single band of 113bp, shows that fluorescent quantitation primer has very strong specificity.By PCR product, send the order-checking of Beijing Qing Kexin industry Bioisystech Co., Ltd, result is consistent with expected results.
(2) establishment of quantitative fluorescent PCR condition
In order to obtain better amplification efficiency and fluorescent signal, according to SuperReal PreMix Plus (SYBR Green) test kit specification sheets, carry out two-step approach and three-step approach is groped, after sample adds, be positioned in ABI7500 quantitative real time PCR Instrument and increase.
Quantitative fluorescent PCR reaction system is: plasmid standard template 1uL, 2 * SuperReal PreMix Plus10uL, each 0.5uL of primer TSWV-113F and TSWV-113R (10umol/L), 50 * ROX Reference Dye5uL, RNase-free ddH
2o3uL, cumulative volume 20uL, mixes rear instantaneous centrifugally, all using the cDNA of healthy Pepper Leaves as template is as negative control at every turn.
Adopt respectively two-step approach and three-step approach to carry out fluorescent quantitative PCR, adopt grads PCR to carry out annealing temperature and grope.
Result shows:
While adopting three-step approach and annealing temperature to be 60 ℃, amplification efficiency is the highest, and specificity is best.
Response procedures after optimization is: 95 ℃ of denaturation 15min, and 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, last 72 ℃ are extended 32s, 40 circulations.
(3) foundation of typical curve
According to formula: the copy number of plasmid (copies/uL)=6.02 * 10
23(copies/mol) * plasmid concentration (g/uL)/plasmid molecule quality (g/mol), draws plasmid copy number.
Through Nano Drop2000c, measuring plasmid standard concentration is 90.2ng/uL, and A260/A280 is 1.98.
Plasmid standard is through 5 times of serial gradients (5
1～5
8doubly), after dilution, copy number is 2.93 * 10
10～3.75 * 10
5copies/uL.Utilize fluorescent quantitation primer TSWV-113F and TSWV-113R, with the quantitative fluorescent PCR reaction conditions of establishing, the plasmid standard after diluting is carried out to absolute quantitation pcr amplification, each extent of dilution arranges 3 repetitions.
Through ABI7500 quantitative real time PCR Instrument software automatic analysis, obtain typical curve, wherein X-coordinate is the logarithmic value (logC) of the initial copy number of serial dilution standard substance template virus, the Ct value (Fig. 4) when ordinate zou is serial dilution standard substance template amplification.
The repeatability of embodiment 4, tomato spotted wilf virus absolute quantitation method and specificity checking
All plant leafs, after extracting total RNA, are inverted to cDNA and quantitatively arrive 1ng/ul.
The cDNA (1ng/ul) of the positive of re-treatment same concentration--TSWV thorn apple blade, the TSWV real time fluorescence quantifying PCR method that adopts embodiment 1 to set up, divide 4 and repeat quantitative fluorescent PCR, obtain amplified fluorescence curve (Fig. 5) and solubility curve (Fig. 6).
As can be seen from Figure 5, the amplified fluorescence curves overlapped of the sample of 4 repetitions together, shows that tomato spotted wilf virus absolute quantitation method of the present invention has good repeatability;
As can be seen from Figure 6, solubility curve is simple spike, and peak value is 80 ℃ of left and right, shows that PCR product is single-minded, does not produce non-specific amplification, and TSWV real time fluorescence quantifying PCR method has good specificity.
The application of embodiment 5, tomato spotted wilf virus absolute quantitation method
Negative control: template is pure water.
Positive control: with the thorn apple blade sample of TSWV.
Plasmid sample: prepare identical plasmid according to the method for embodiment 2, measuring plasmid sample starting point concentration through Nano Drop2000c is 6.87ng/ul, and (dilution range is from 5 with 5 times of gradient stepwise dilution liquid of DEPC water
4doubly to 5
8doubly, according to formula: the copy number of plasmid (copies/uL)=6.02 * 10
23(copies/mol) * plasmid concentration (g/uL)/plasmid molecule quality (g/mol), under each weaker concn, the reason copy number of the middle goal gene of plasmid sample is respectively: 3.25 * 10
8, 6.5 * 10
7, 1.3 * 10
7, 2.6 * 10
6, 5.8 * 10
5.
Three parts of doubtful Pepper Leaves samples with TSWV (L1, L2, L3): random acquisition.
The typical curve (y=-3.4581x+43.701) of the plasmid standard of setting up according to the method for embodiment 3, TSWV content in negative control, positive control sample, 5 parts, plasmid sample and the doubtful Pepper Leaves sample L1 with TSWV, L2, L3 is calculated, and result shows below table:
| Sample | Ct value | Goal gene copy number | Default copy number |
| Negative control | 38.35 | 35.27 | 0 |
| Positive control | 16.25 | 8.67×10 7 | ? |
| Plasmid diluted sample 5 4Doubly | 14.27 | 3.24×10 8 | 3.25×10 8 |
| Plasmid diluted sample 5 5Doubly | 16.69 | 6.47×10 7 | 6.50×10 7 |
| Plasmid diluted sample 5 6Doubly | 19.12 | 1.28×10 7 | 1.30×10 7 |
| Plasmid diluted sample 5 7Doubly | 21.52 | 2.59×10 6 | 2.60×10 6 |
| Plasmid diluted sample 5 8Doubly | 23.94 | 5.81×10 5 | 5.80×10 5 |
| L1 | 23.80 | 5.69×10 5 | ? |
| L2 | 18.75 | 1.64×10 7 | ? |
| L3 | 21.51 | 2.61×10 6 | ? |
Note: after Ct value surpasses 35, fluorescent quantitation Ct value approaches negative control, data reliability needs further to be confirmed.
As can be seen from the above table, the goal gene copy number detecting under five extension rates of synthetic cDNA sample conforms to the copy number of calculating by Nano Drop2000c mensuration concentration, without significant difference, prove that method of the present invention can accurate quantitative analysis goal gene copy number.
Claims (8)
1. the plant sample to be measured of doubtful infection tomato spotted wilf virus is carried out to a method for spotted wilt virus absolute quantitation, comprise the steps:
(1) prepare spotted wilt virus plasmid standard typical curve, step is as follows:
To determine the positive sample of plant that infects tomato spotted wilf virus, extract total RNA, reverse transcription becomes positive cDNA; Take positive cDNA as template, with tomato spotted wilf virus Auele Specific Primer TSWV-482F/TSWV-482R amplification tomato spotted wilf virus specific fragment; Reclaim this specific fragment and be cloned in conventional skeleton carrier and obtain plasmid standard;
Survey the concentration of plasmid standard, according to the copy number of plasmid (copies/uL)=6.02 * 10
23(copies/mol) * plasmid concentration (g/uL)/plasmid molecule quality (g/mol), draws the viral copy number of plasmid standard;
Gradient ground dilution plasmid standard, with fluorescent quantitation primer, TSWV-113F/TSWV-113R carries out quantitative fluorescent PCR to the plasmid standard of different concns, draws the Ct value of each concentration plasmid standard;
The logarithmic value of viral copy number corresponding to each concentration plasmid standard of take is X-coordinate, take Ct value as ordinate zou Criterion curve;
Described tomato spotted wilf virus Auele Specific Primer TSWV-482F/TSWV-482R, sequence is:
5’-TCTGGTAGCATTCAACTTCA-3’，
5’-CTTCTCTGGTGTCATACTTCT-3’；
The sequence of described fluorescent quantitation primer TSWV-113F/TSWV-113R is:
5’-CTTGCCATAATGCTGGGAGGTAG-3’/5’-TCCCGAGGTCTTTGTATTTTGC-3’；
(2) extract total RNA of described plant sample to be measured, cDNA is synthesized in reverse transcription; And take cDNA as template, TSWV-113F/TSWV-113R primer carry out quantitative fluorescent PCR, obtain the Ct value of plant sample to be measured; Described Ct is brought into the copy number that calculates tomato spotted wilf virus in plant sample to be measured in described typical curve.
2. method according to claim 1, the system of described quantitative fluorescent PCR is: template 1uL, 2 * SuperReal PreMix Plus10uL, each 0.5uL of 10umol/L fluorescent quantitation primer TSWV-113F/TSWV-113R, 50 * ROX Reference Dye5uL, RNase-free ddH
2o3uL, cumulative volume 20uL.
3. method according to claim 2, the reaction conditions of described quantitative fluorescent PCR is: 95 ℃ of denaturation 15min, 95 ℃ of sex change 10s, 60 ℃ of annealing 32s, 72 ℃ are extended 32s, 40 circulations.
4. method according to claim 1, the concentration of described survey plasmid standard adopts Nano Drop2000c.
5. method according to claim 1, described conventional skeleton carrier refers to pMD
tM18-T.
6. the application of the arbitrary described method of claim 1～5 in tomato spotted wilf virus absolute quantitation.
7. for detection of the specific PCR primer of tomato spotted wilf virus, its nucleotides sequence is classified as:
TSWV-482F?5’-TCTGGTAGCATTCAACTTCA-3’；
TSWV-482R?5’-CTTCTCTGGTGTCATACTTCT-3’。
8. for detection of testing sample, whether carry the method for tomato spotted wilf virus, comprise the steps:
(1) extract the RNA of testing sample, cDNA (quantitatively arriving 1ng/ul) is synthesized in reverse transcription;
(2) take the synthetic cDNA of reverse transcription carries out PCR reaction as template, obtains amplified production; The primer of PCR reaction is TSWV-482F/TSWV-482R:
5’-TCTGGTAGCATTCAACTTCA-3’/5’-CTTCTCTGGTGTCATACTTCT-3’；
(3) agarose gel electrophoresis detects amplified production, if the size of amplified production is 482bp, in testing sample, carries tomato spotted wilf virus; If the size of amplified production is not 482bp, in testing sample, do not carry tomato spotted wilf virus;
The system of described PCR reaction is: cDNA template 2uL, TSWV-482F0.5uL, TSWV-482R0.5uL, 2 * ES Tap MasterMix10uL, ddH
2o7uL, cumulative volume 20uL;
The condition of described PCR reaction is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ of extension 45s, 40 circulations, last 72 ℃ are extended 5min.
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| CN104404173A (en) * | 2014-12-21 | 2015-03-11 | 山东出入境检验检疫局检验检疫技术中心 | NASBA method for detecting tomato spotted wilt virus |
| CN104450975A (en) * | 2014-12-31 | 2015-03-25 | 山东出入境检验检疫局检验检疫技术中心 | RT-HDA primer, kit and method for detecting tomato spotted wilt virus |
| CN104498632A (en) * | 2014-12-25 | 2015-04-08 | 山东出入境检验检疫局检验检疫技术中心 | Method for detecting tomato spotted wilf virus based on loop-mediated isothermal amplification technology |
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| CN112625141A (en) * | 2020-12-28 | 2021-04-09 | 昆明海关技术中心 | Protein standard substance of tomato spotted wilt virus and application thereof |
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| CN115820930A (en) * | 2022-09-26 | 2023-03-21 | 云南省农业科学院生物技术与种质资源研究所 | RT-qPCR detection method for single tomato seed carrying tomato spotted wilt virus TSWV and application thereof |
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