CN105018612A - Quantitative detection method of garlic virus - Google Patents
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Abstract
The invention discloses a quantitative detection method of a garlic virus, relates to virus detection methods and belongs to the technical field of plant virology. The quantitative detection method includes the steps of extraction of total RNA of garlic, primer designing and synthesizing, preparation of a reverse transcription product cDNA template, PCR amplification and recovery, competent cell preparation, recombinant plasmid transformation, standard curve making and virus detection and calculation. By applying the quantitative detection method, existence of the virus can be detected qualitatively, and accumulation quantity of the virus can be quantified quickly and accurately. The method is conducive to prevention and control of garlic virus diseases and plays an active role in improving yield and quality of garlic.
Description
Technical field
The present invention relates to the detection method of Garlic Virus, belong to plant virology technical field, specifically a kind of quantitative detecting method of Garlic Virus.
Background technology
Garlic (Allium sativum L.) is Liliaceae allium 1 year or biennial herb plant, eat as tradition and have a long history with medicinal, it contains abundant nutritive substance, such as VITAMIN, amino acid, trace element-selenium and germanium, polysaccharide etc., especially containing unique active substance---alliin.Medical research confirms, garlic have significantly antibacterial, prevention and therapy cardiovascular disorder, reduce blood sugar, many-sided clinical effectiveness such as anticancer, antitumor.In recent years, along with the increase gradually of international and domestic garlic demand, garlic cultivated area worldwide is also expanding thereupon.Usual garlic carries out vegetative propagation with bulb, and long-term vegetative propagation makes the virus of garlic In vivo infection transmit by generation and to accumulate, and causes virus disease to increase the weight of year by year and garlic kind sexual involution, production makes output reduce by 30% ~ 60%.
Research finds, cause the cause of disease kind of Garlic Virus disease more, wherein OYDV (the Onion yellow dwarf virus of Potyvirus, and Leek yellow stripe virus (Leek yellowstripe virus OYDV), LYSV) two-strain is the main virus of harm Garlic yield and quality, and in garlic plant, infection rate is respectively higher than 50% and 20%.Detection method for Garlic Virus is also existing at present much studies report, and concrete detection method has: outward appearance symptomatic diagnosis method, biological characteristis (plant indicator differential method), electron microscope method, determination of serology method, mass spectrometry, Protocols in Molecular Biology.The principle of molecular Biological Detection is according to the detection method based on viral RNA, current method for measuring mainly contains: dsRNA electrophoretic technique, polymerase chain reaction technology, hybridization, Real-Time Fluorescent Quantitative PCR Technique (quantitative real-time polymerase chain reaction, qRT-PCR) etc.The principle of qRT-PCR first plant is carried out to the extraction of total serum IgE, then under the effect of viral special primer, virus mRNA reverse transcription is become cDNA, fluorophor is added in PCR reaction system, fluorescent signal is utilized to accumulate the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template is carried out to the method for quantitative analysis, the method have highly sensitive, high specificity, detection speed are fast, the high many-sided advantage of accuracy.Because plant virus more than 70% is RNA viruses, so qRT-PCR is most widely used in plant virus detects.Therefore, being necessary to set up qRT-PCR detection method fast and effectively for garlic OYDV and LYSV virus, is the prevention and control of Garlic Virus disease, and the yield and quality improving garlic provides technical support.
Summary of the invention
The object of the invention is the quantitative detecting method proposing a kind of Garlic Virus, for garlic OYDV and LYSV virus provide one qRT-PCR detection method fast and accurately.
For achieving the above object, the quantitative detecting method of a kind of Garlic Virus of the present invention, comprising: the extraction of garlic yellow dwarf virus total serum IgE, prepare the detection of reverse transcription product cDNA template, the amplification of goal gene fragment, the recovery purifying of PCR fragment, the structure of recombinant plasmid, competent cell preparation, the conversion of recombinant plasmid, the extraction of plasmid DNA, the making of typical curve and virus; The amplification of described goal gene fragment for template, utilizes the Auele Specific Primer of OYDV virus to carry out pcr amplification to sample with reverse transcription product cDNA; Reaction conditions is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s; 55 DEG C of annealing 30s; 72 DEG C extend 1min; 33 circulations; 72 DEG C extend 10min; 4 DEG C of termination reactions.
Standard substance are carried out 10 times of dilutions by the making of described typical curve: with the linearizing plasmid DNA containing goal gene for standard substance successively, calculate copy number,
Copy number=(plasmid concentration (ng/ μ L) × 10-9 (g) × 6.023 × 1023)/660 × Plasmid Base number,
Reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, gathers fluorescent signal, detect in real time at the end of the annealing temperature of each circulation.Each template concentrations does 3 repetitions, averages for interpretation of result.
Complete in the steps below:
(A) garlic total serum IgE is extracted:
1) polysaccharide polyphenol total RNA extraction reagent box is adopted to carry out extraction and isolation to RNA in garlic blade;
2) adopt the quality of spectrophotometry RNA, require OD
260/ OD
280ratio 2 ~ 2.1;
3) 1.0% sepharose is utilized to detect the quality of RNA;
(B) design of primers and synthesis:
According to the viral nucleotide sequences accession number (OYDV:AJ292223 that GenBank has reported, LYSV:AY0007693), NCBI BLAST system is utilized to carry out tetraploid rice, select the conserved sequence of two-strain coat protein, Primer Premier5.0 is utilized to carry out design of primers, the primer sequence (227bp) of OYDV is: upstream 5 '-AGTGATGCAGCTGAAGCATACATT-3 ', downstream 5 '-ACGTTACCATCCAGGCCAAA-3 '; The primer sequence (66bp) of LYSV is: upstream 5 '-TCTCGCACGGTATGCATTTG-3 ', downstream 5 '-GCCTCGCGCGCTCTAA-3 ';
(C) reverse transcription product cDNA template is prepared: adopt Reverse Transcription box to prepare cDNA, response procedures is: 37 DEG C, 15min, 85 DEG C, 5sec;
(D) pcr amplification and recovery:
1) pcr amplification test kit is adopted to carry out the amplification of goal gene fragment;
2) OYDV response procedures is: 94 DEG C of denaturation 2min, and then 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, circulate 35 times, and last 72 DEG C extend 5min;
3) LYSV response procedures is: 94 DEG C of denaturation 5min, and then 94 DEG C of 1min, 53 DEG C of 1min, 72 DEG C of 1.5min, circulate 40 times, and last 72 DEG C extend 10min;
4) adopt PCR primer purifying to reclaim test kit and reclaim purifying object fragment;
(E) competent cell is prepared:
1) coli strain of-70 DEG C of freezen protective is applied on LB solid plate substratum, 37 DEG C of incubated overnight;
2) picking list bacterium colony puts into 20mL LB liquid nutrient medium, 220rpm, 37 DEG C of cultivation 16h;
3) in the ratio of 1:100, the bacterium liquid of above-mentioned cultivation is proceeded in 100mL LB liquid nutrient medium, 220rpm, 37 DEG C be cultured to OD
6000.3 ~ 0.6;
4) the bacterium liquid of cultivation is placed in ice bath 30min, 5000rpm, 4 DEG C of centrifugal 5min, remove supernatant liquor, precipitation be placed in ice bath;
5) precipitation is resuspended in the 0.1mol/L MgCl of 40mL precooling
2in solution, in ice bath, 10min, 5000rpm, 4 DEG C of centrifugal 5min, abandon supernatant;
6) precipitation is resuspended in 1mL 0.1mol/L CaCl
2(glycerine containing 10%), 20min in ice bath;
7) by often pipe 100 μ L packing, in-70 DEG C of preservations, prepare competent quality by transforming known plasmid inspection simultaneously.
(F) recombinant plasmid is transformed:
1) preparation is containing the LB solid medium of Ampicillin Trihydrate (Ampicill in, Amp), and adds respectively after 100 μ LX-Gal and 100 μ L IPTG smoothen place 30min in dark;
2) get-70 DEG C of competent escherichia coli cells preserved, slowly melt in ice bath, 10 μ L are connected product and adds in competent cell, mixing, ice bath 30min;
3) take out centrifuge tube, be placed in water-bath 42 DEG C of heat shock 90sec, period does not rock centrifuge tube, rear rapid ice bath 5min;
4) add 400 μ L LB liquid nutrient mediums in each centrifuge tube, piping and druming is even, 180rpm, 37 DEG C of shaking culture 1h;
5) get 200 μ L bacterium liquid, be spread evenly across on the LB solid medium got ready, room temperature places 30min, after solution is completely absorbed, is inverted for 37 DEG C and cultivates 16h;
6) positive and negative control are set, guarantee the accuracy of test-results;
(G) production standard curve:
1) measure the plasmid DNA concentration being loaded with OYDV fragment and LYSV fragment respectively, concentration calculates the copy number of two kinds of plasmid DNA respectively accordingly, original copy number is diluted 10 times successively by the method for gradient dilution, obtains 10 respectively
8, 10
7, 10
6, 10
5, 10
4five copy numbers, adopt fluorescence quantitative kit to carry out the quantitative of copy number; 2) take plasmid copy number as X-coordinate, C
tvalue obtains typical curve for ordinate zou, and the typical curve of OYDV is C
t=-3.139 × log (X)+38.62, R
2=0.995; The typical curve of LYSV is C
t=-3.454 × log (X)+39.13, R
2=0.985;
(H) according to OYDV in garlic blade at 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations; LYSV at 94 DEG C of denaturation 10min, 95 DEG C of sex change 16s, 60 DEG C of annealing/extension 32s, 38 circulations; Obtain corresponding C respectively
tvalue, substitutes into respective standard curve in (H), can obtain the accurate copy number of corresponding virus, thus the quantitative expression amount determining Garlic Virus.
The quantitative detecting method of a kind of Garlic Virus of the present invention, its beneficial effect is: apply qRT-PCR technology for detection garlic OYDV of the present invention and LYSV two-strain, have fast the Garlic Virus of field Combined Infection, save time, advantage that accuracy is high, not only can detect qualitatively virus existence, the more important thing is can be quantitative the content detecting Garlic Virus.The foundation of this method is not only conducive to the prevention and control of Garlic Virus disease, also has simultaneously act on energetically the yield and quality aspect improving garlic.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only one of them embodiment of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is total serum IgE electrophorogram;
Fig. 2 is LYSV and OYDV reverse transcription PCR amplification figure;
Fig. 3 is OYDV typical curve;
Fig. 4 is OYDV amplification curve;
Fig. 5 is OYDV solubility curve;
Fig. 6 is LYSV typical curve;
Fig. 7 is LYSV amplification curve;
Fig. 8 is LYSV solubility curve.
Embodiment
Embodiment 1
A quantitative detecting method for Garlic Virus, completes in the steps below:
1. the extraction of garlic total serum IgE: 1) " folk music purple garlic " fresh garlic blade of clip Gansu Province Minyue County's field planting, Liquid nitrogen storage, take back laboratory for subsequent use, adopt polysaccharide polyphenol total RNA extraction reagent box to carry out extraction and isolation to RNA in garlic blade.Concrete grammar is: 1. sample preparation: get garlic blade 0.2g, fully grind in liquid nitrogen, and every 50 ~ 100mg tissue adds 1mL lysate RZ, carries out homogenized with Syrup-homogenizing instrument; 2. homogenised sample is placed 5min at 20 DEG C, make to adjust albumen composition and be separated completely; 3. 4 DEG C of centrifugal 5min of 12000rpm, get supernatant, proceed to one new in the centrifuge tube of RNase; 4. add 200 μ L chloroforms, build pipe lid, concuss 15s, room temperature places 3min; 5. 4 DEG C of centrifugal 10min of 12000rpm, sample can be divided into three layers: yellow organic phase, middle layer and colourless aqueous phase, and RNA, mainly in aqueous phase, transfers to aqueous phase in new pipe, carries out next step operation; 6. 0.5 times of volume dehydrated alcohol is slowly added, mixing (now may occur precipitation).To obtain solution proceeds in adsorption column together with precipitation, and 4 DEG C of centrifugal 30sec of 12000rpm, discard the waste liquid in collection tube; 7. in adsorption column, add 500 μ L protein liquid removals, 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; 8. in adsorption column, add 700 μ L rinsing liquids, room temperature leaves standstill 2min, and 4 DEG C of centrifugal 30sec of 12000rpm, abandon waste liquid; This step of repetitive operation once; 9. adsorption column is put into 2mL collection tube, 4 DEG C of centrifugal 2min of 12000rpm, remove residual liquid.Be placed in ventilating kitchen and ventilate a moment, fully to dry; 10. adsorption column is proceeded in a new 1.5mL centrifuge tube, add 50 μ L RNase-Free ddH
2o, room temperature places 2min, and 4 DEG C of centrifugal 2min of 12000rpm, products therefrom is in-70 DEG C of preservations.2) total serum IgE of extraction is drawn 2 μ L, use RNase-free H
2o dilutes 100 times, is determined at the light absorption value under 260nm and 280nm, requires OD with spectrophotometer TU-1810
260/ OD
280ratio 2 ~ 2.1; 3) 4.5 μ LRNA samples are drawn, add 2 μ L 5 × MOPS, 3.5 μ L formaldehyde, 10 μ L deionized formamides, 65 DEG C, water-bath 10min, add a small amount of EB and the rear point sample of tetrabromophenol sulfonphthalein mixing again, electrophoresis (80V) 1.5h in 1.0% sepharose, requires in ultraviolet detection that 28S and 18S band is bright, clear, strip edge is clear, if the brightness of 28S band is that more than the twice of 18S is for best.
2. design of primers and synthesis: the viral nucleotide sequences accession number (OYDV:AJ292223 reported according to GenBank, LYSV:AY0007693), NCBI BLAST system is utilized to carry out tetraploid rice, select the conserved sequence of two-strain coat protein, Primer Premier5.0 is utilized to carry out design of primers, the primer sequence (227bp) of OYDV is: upstream 5 '-AGTGATGCAGCTGAAGCATACATT-3 ', downstream 5 '-ACGTTACCATCCAGGCCAAA-3 '; The primer sequence (66bp) of LYSV is: upstream 5 '-TCTCGCACGGTATGCATTTG-3 ', downstream 5 '-GCCTCGCGCGCTCTAA-3 ', transfers to biotech firm to synthesize the primer designed.
3. prepare reverse transcription product cDNA template: adopt Reverse Transcription box to carry out the synthesis of cDNA, reaction system is as following table, and reverse transcription condition is: 37 DEG C, 15min, 85 DEG C, 5sec; Of short duration centrifugal after mixing gently, in 37 DEG C of water-bath 15min, 85 DEG C of water-bath 5s termination reactions, are stored in 4 DEG C by products therefrom.
4. the amplification of goal gene fragment: with above-mentioned reverse transcription product cDNA for template, utilizes the Auele Specific Primer of OYDV and LYSV virus to carry out pcr amplification to sample.As following table, OYDV response procedures is reaction system (50 μ L): 94 DEG C of denaturation 2min, and then 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, circulate 35 times, and last 72 DEG C extend 5min; 3) LYSV response procedures is: 94 DEG C of denaturation 5min, and then 94 DEG C of 1min, 53 DEG C of 1min, 72 DEG C of 1.5min, circulate 40 times, and last 72 DEG C extend 10min.
The recovery purifying of 5.PCR fragment: use DNA fragmentation to reclaim test kit fast and reclaim object fragment; Concrete steps are as follows: 1) to adsorption column CA
2in add 500 μ L balance liquid BL, the centrifugal 2min of 12000rpm, outwells the waste liquid in collection tube, is reentered into by adsorption column in collection tube; 2) the DNA band of the expection size fragment on sepharose cuts with clean blade after completing by electrophoresis, is transferred in centrifuge tube; 3) added by the sol solutions PN of blob of viscose volume 3 times and hold in the centrifuge tube of blob of viscose, 50 DEG C of water-baths place 10min, and blob of viscose is dissolved completely; Taking out centrifuge tube makes solution temperature be down to room temperature; 4) above-mentioned solution is added CA
2in, by CA
2put into collection tube, the centrifugal 2min of 12000rpm after room temperature placement 2min, abandons waste liquid, again by CA
2put into collection tube; 5) rinsing liquid PW is to adsorption column CA
2in add 600uL, the centrifugal 2min of 12000rpm, outwells waste liquid, by CA
2put into collection tube; Rinsing liquid PW adds 600 μ L again, and the centrifugal 2min of 12000rpm, outwells waste liquid; 6) collection tube puts into CA
2, the centrifugal 2min of 12000rpm, to eliminate rinsing liquid as far as possible, rear room temperature leaves standstill 5min, thoroughly dries; 7) new centrifuge tube puts into CA
2, elution buffer EB drips 40 μ L in film mid-way, and room temperature leaves standstill 10min.The centrifugal 2min of 12000rpm, eluted dna.
6. the structure of recombinant plasmid: select the product reclaimed through rubber tapping to be used for ligation.Select PMD18-T cloning vector to connect, reaction system is as following table, and mixed system gently, in water-bath, 16 DEG C are reacted 2min.
7. prepare competent cell: 1) be applied to by the coli strain of-70 DEG C of freezen protective on LB solid plate substratum, 37 DEG C of incubated overnight; 2) picking list bacterium colony puts into 20mL LB liquid nutrient medium, 220rpm, 37 DEG C of cultivation 16h; 3) in the ratio of 1:100, the bacterium liquid of above-mentioned cultivation is proceeded in 100mL LB liquid nutrient medium, 220rpm, 37 DEG C be cultured to OD
6000.5; 4) the bacterium liquid of cultivation is placed in ice bath 30min, 5000rpm, 4 DEG C of centrifugal 5min, remove supernatant liquor, precipitation be placed in ice bath; 5) precipitation is resuspended in the 0.1mol/L MgCl of 40mL precooling
2in solution, in ice bath, 10min, 5000rpm, 4 DEG C of centrifugal 5min, abandon supernatant; 6) precipitation is resuspended in 1mL 0.1mol/L CaCl
2(glycerine containing 10%), 20min in ice bath; 7) by often pipe 100 μ L packing, in-70 DEG C of preservations, prepare competent quality by transforming known plasmid inspection simultaneously.
8. transform recombinant plasmid: 1) preparation is containing the LB solid medium of Ampicillin Trihydrate (Amp), and add respectively after 100 μ L X-Gal and 100 μ L IPTG smoothen place 30min in dark; 2) get-70 DEG C of competent escherichia coli cells preserved, slowly melt in ice bath, 10 μ L are connected product and adds in competent cell, mixing, ice bath 30min; 3) take out centrifuge tube, be placed in water-bath 42 DEG C of heat shock 90sec, period does not rock centrifuge tube, rear rapid ice bath 5min; 4) add 400 μ L LB liquid nutrient mediums in each centrifuge tube, piping and druming is even, 180rpm, 37 DEG C of shaking culture 1h; 5) get 200 μ L bacterium liquid, be spread evenly across on the LB solid medium got ready, room temperature places 30min, after solution is completely absorbed, is inverted for 37 DEG C and cultivates 16h; 6) positive and negative control are set, guarantee the accuracy of test-results.
9. the qualification of recombinant plasmid: the white list bacterium colony 1) on picking LB flat board, carry out pcr amplification thus qualification recombinant plasmid, PCR reaction system is as following table; 2) according to the cloning site at target DNA fragment restriction enzyme site analytical results and PMD18-T carrier two ends, choice for use SacI, Hind III, Sal I restriction enzyme, carry out double digestion qualification to recombinant plasmid; 3) double digestion reaction system is: each 1 μ L of restriction enzyme, damping fluid 2 μ L, plasmid DNA 2 μ L, ddH
2o 4 μ L, reacts 6h in 37 DEG C of water-baths; 4) checked order by biotech firm after the positive bacterium colony of qualification extracts plasmid, the virus sequence reported in sequencing result and ncbi database carries out tetraploid rice analysis.
10. the extraction of plasmid DNA: use plasmid DNA purification test kit, concrete steps are as follows: 1) colibacillary cultivation: from plate culture medium picking individual colonies be seeded to 2mL containing in antibiotic liquid nutrient medium, 37 DEG C of incubated overnight 16h; 2) get the incubated overnight bacterium liquid of 2mL, the centrifugal 2min of 12000rpm, abandons supernatant; 3) add 250 μ L solution I, abundant suspended bacterial precipitation, room temperature places 10-15min; 4) solution II adding 250 μ L spins upside down mixing 5 ~ 6 times gently, makes the abundant cracking of thalline, forms clear solution; 5) add in centrifuge tube by the solution III of 350 μ L4 DEG C precoolings, spin upside down mixing gently 5 ~ 6 times, until form consolidation aggegation block, room temperature leaves standstill 2min, the centrifugal 10min of room temperature 12000rpm; 6) drawing supernatant proceeds in post, and the centrifugal 1min of 12000rpm, abandons waste liquid; 7) add the Buffer WA of 500 μ L, the centrifugal 30s of 12000rpm, abandons waste liquid; 8) add the Buffer WB of 700 μ L, the centrifugal 30s of 12000rpm, abandons waste liquid, repeats this step once; 9) the centrifugal 1min of 12000rpm, eliminates residual washing lotion; 10) be placed in by post on the centrifuge tube of new 1.5ml, add 50 μ L sterile purified waters in the centre of film, room temperature leaves standstill 1min, the centrifugal 1min of 12000rpm, and eluted dna ,-20 DEG C save backup.
11. production standard curves: 1) with the linearizing plasmid DNA containing goal gene for standard substance, standard substance are carried out successively 10 times of dilutions, obtain 10 respectively
8, 10
7, 10
6, 10
5, 10
4five copy numbers, calculate copy number, copy number=(plasmid concentration (ng/ μ L) × 10
-9(g) × 6.023 × 10
23)/660 × Plasmid Base number; If blank, using different concns standard substance as masterplate, carry out qRT-PCR, response procedures is as following table; 2) response procedures of OYDV is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations; The response procedures of LYSV is: 94 DEG C of denaturation 10min, 95 DEG C of sex change 16s, 60 DEG C of annealing/extension 32s, 38 circulations; 3) each template concentrations does 3 repetitions, averages for interpretation of result; 4) take plasmid copy number as X-coordinate, C
tvalue obtains typical curve for ordinate zou, and the typical curve of OYDV is C
t=-3.139 × log (X)+38.62, R
2=0.995; The typical curve of LYSV is C
t=-3.454 × log (X)+39.13, R
2=0.985.
The detection of 12. viruses and calculating: according to Methods and steps in above-mentioned 1 ~ 11, the extraction of " folk music purple garlic " fresh garlic blade total serum IgE of Minyue County of Gansu Province field planting, reverse transcription cDNA, the amplification of goal gene fragment, the recovery purifying of PCR fragment and qRT-PCR are detected, finally obtain the average C of OYDV and LYSV
tvalue, is respectively 16.70 and 15.43, brings formula in 11 into and can obtain the copy number of OYDV and LYSV in garlic blade and be respectively 9.62 × 10
6with 7.41 × 10
6.
Claims (5)
1. a quantitative detecting method for Garlic Virus, the method comprises: the extraction of garlic yellow dwarf virus total serum IgE, prepare the detection of reverse transcription product cDNA template, the amplification of goal gene fragment, the recovery purifying of PCR fragment, the structure of recombinant plasmid, competent cell preparation, the conversion of recombinant plasmid, the extraction of plasmid DNA, the making of typical curve and virus; It is characterized in that: the amplification of described goal gene fragment for template, utilizes the Auele Specific Primer of OYDV virus to carry out pcr amplification to sample with reverse transcription product cDNA; Reaction conditions is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s; 55 DEG C of annealing 30s; 72 DEG C extend 1min; 33 circulations; 72 DEG C extend 10min; 4 DEG C of termination reactions.
2. the quantitative detecting method of a kind of Garlic Virus as claimed in claim 1, is characterized in that: the making of described typical curve: with the linearizing plasmid DNA containing goal gene for standard substance, standard substance is carried out successively 10 times of dilutions, calculates copy number,
Copy number=(plasmid concentration (ng/ μ L) × 10-9 (g) × 6.023 × 1023)/660 × Plasmid Base number,
Reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, gathers fluorescent signal, detect in real time at the end of the annealing temperature of each circulation; Each template concentrations does 3 repetitions, averages for interpretation of result.
3. the quantitative detecting method of a kind of Garlic Virus as claimed in claim 1, it is characterized in that: the method for the detection of described virus is: extract garlic blade RNA, reverse transcription is cDNA, take cDNA as template, carries out real-time fluorescence quantitative PCR, reaction system is as follows: reaction conditions is: 95 DEG C of denaturation 10min, 95 DEG C of sex change 15s, 60 DEG C of annealing/extension 30s, 40 circulations, at the end of the annealing temperature of each circulation, gather fluorescent signal, detect in real time.
4. the quantitative detecting method of a kind of Garlic Virus as claimed in claim 3, is characterized in that: described competent cell preparation:
(1) by-70 DEG C of frozen coli strain setting-outs on LB solid plate, 37 DEG C of incubated overnight;
(2) picking list bacterium colony puts into 20mL LB liquid nutrient medium, 220rpm, cultivates about 16hr for 37 DEG C;
(3) in the ratio of 1:100, proceeded to by the bacterium liquid of above-mentioned cultivation in 100mL LB liquid nutrient medium, 220rpm, 37 DEG C are cultured to OD6000.3 ~ 0.6;
(4) the bacterium liquid of cultivation is put in ice bath 30min in ice;
(5) 5000rpm, 4 DEG C of centrifugal 5min, remove supernatant liquor, precipitation are put on ice;
(6) precipitation is resuspended in the 0.1M MgCl of 40ml precooling
2;
(7) 10min is on ice placed in;
(8) 5000rpm, 4 DEG C of centrifugal 5min, abandon supernatant;
(9) precipitation is resuspended in the 0.1M CaCl of 1ml ice bath
2(glycerine containing 10%), places 10 ~ 30min on ice;
(10) by often pipe 100 μ L packing, in-70 DEG C of preservations, prepare competent quality by transforming known plasmid inspection simultaneously.
5. the quantitative detecting method of a kind of Garlic Virus as claimed in claim 1, is characterized in that: the conversion of described recombinant plasmid:
(1) preparation is containing the LB solid medium of Amp, and adds respectively after 100 μ L X-Gal and 100 μ L IPTG smoothen place 30min in dark;
(2) get-70 DEG C of competent escherichia coli cells preserved, in slowly melting on ice, afterwards 10 μ l being connected product and adding in competent cell, mixing, ice bath 30min;
(3) take out centrifuge tube, be placed in water-bath 42 DEG C of heat shock 90sec, period does not rock centrifuge tube.Rear ice bath 5min rapidly;
(4) add 400 μ L LB liquid nutrient mediums in each centrifuge tube, piping and druming is even, 37 DEG C of 180rpm shaking culture 1h;
(5) get 200 μ l bacterium liquid, be spread evenly across on the LB solid medium got ready, room temperature places 30min, after solution is completely absorbed, is inverted cultivation 12 ~ 16h for 37 DEG C;
(6) positive and negative control are set, guarantee the accuracy of test-results.
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| CN107541506A (en) * | 2016-06-23 | 2018-01-05 | 新疆农业大学 | A kind of Xinjiang garlic wild relatives DNA extracting method |
| CN110283945A (en) * | 2019-07-26 | 2019-09-27 | 中国农业科学院蔬菜花卉研究所 | Garlic Virus detection method and primer |
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| CN103911461A (en) * | 2014-03-13 | 2014-07-09 | 湖南农业大学 | A method of simultaneously detecting a plurality of garlic viruses by quadruple RT-PCR and a primer composition thereof |
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| CN107541506A (en) * | 2016-06-23 | 2018-01-05 | 新疆农业大学 | A kind of Xinjiang garlic wild relatives DNA extracting method |
| CN107541506B (en) * | 2016-06-23 | 2021-04-09 | 新疆农业大学 | Method for extracting DNA of wild allied species of Xinjiang garlic |
| CN107447050A (en) * | 2017-09-08 | 2017-12-08 | 南京农业大学 | A kind of method that comprehensive quick detection garlic RNA virus is sequenced using transcript profile |
| CN107447050B (en) * | 2017-09-08 | 2020-11-24 | 南京农业大学 | Method for comprehensively and rapidly detecting garlic RNA virus by using transcriptome sequencing |
| CN110283945A (en) * | 2019-07-26 | 2019-09-27 | 中国农业科学院蔬菜花卉研究所 | Garlic Virus detection method and primer |
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