CN103484567B - Narcissus late season yellows virus detection kit and method - Google Patents
Narcissus late season yellows virus detection kit and method Download PDFInfo
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Abstract
The invention relates to a narcissus late season yellows virus detection kit and method specific for narcissus late season yellows virus detection. The knit comprises an outside upstream primer, an outside downstream primer, an inside upstream primer, an inside downstream primer, an RTBuffer, an RNA enzyme inhibiting factor, reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, TaqDNA polymeras, positive control, negative control and RNase-free ddH2O. Specific primers are designed according to narcissus late season yellows virus gene sequences, and a nested RT-PCR technology is utilized to detect narcissus late season yellows viruses. Firstly, inverse transcription is carried out with extracted RNA as the template to synthesize cDNA, first-round PCR amplification is carried out with the cDNA as the template, second-round PCR amplification is then carried out with first-round PCR products as the template, and agarose gel electrophoresis detection is conducted on amplification products: if a specificity target fragment of the size of 539bp appear, the judgment result indicates that a detected virus is a narcissus late season yellows virus. The narcissus late season yellows virus detection kit and method have the advantages of being strong in specificity, high in sensitivity, good in accuracy and short in detection time and are suitable for quickly detecting and monitoring narcissus late season yellows viruses in entry ports, exit ports and agricultural production.
Description
Technical field
The present invention relates to a kind of narcissus slow season yellow virus detection kit and detection method thereof, belong to Plant Quarantine technical field, be applicable to pass in and out and agriculture production on rapid detection and the monitoring of the slow season yellow of narcissus virus.
Background technology
The slow season yellow of narcissus virus (Narcissus late season yellows virus, NLSYV) belong to marmor upsilon section (Potyviridae) Potyvirus (Potyvirus) member, this virus has the characteristic similar with other marmor upsilon, in infected narcissus cell, there will be typical pinwheel inclusion.Viral genome is sense single stranded rna, total length approximately 9,651bp.The slow season yellow of narcissus virus (NLSYV) was in the news as far back as 1980, and after this people have carried out relevant research to its virion structure, host range, hazard symptoms, circulation way and serological characteristic etc.NLSYV plastochondria form is linear, big or small about 750nm × 12nm.NLSYV natural host is narrower, some kinds that are mainly confined to narcissus of having reported.NLSYV infects after narcissus and can cause on blade, scape and occur that the long narrow symptom such as chlorisis striped or ellipticity patch, narcissus diseased plant are normally not present deformity, downgrade symptom.Symptom occurs in the week of the 2-3 after the florescence greatly, plant old and feeble withering gradually subsequently, but this disease symptom not necessarily all occurs every year.NLSYV can be by there being wing black peach aphid (Myzus persicae) amboceptor to propagate between narcissus plant.NLSYV on serological relation with marmor upsilon (Potato virus Y, PVY), Brassica 2 et 4 (Turnip mosaic virus, TuMV) and six go out colored mosaic virus (Alstroemeria mosaic virus, AIMV) there is serology dependency, but there is no serological relation with another narcissus degeneration virus (Narcissus degeneration virus, NDV) that is all potyvirus.It is reported, NLSYV can also with other plant virus Combined Infection narcissus, for example Herba Phyllanthi Urinariae's mosaic virus (Ornithogalum mosaic virus, OrMV), narcissus symptomless virus (Narcissus symptomless virus, NSV).NLSYV infects after narcissus, can affect the quality of narcissus commodity, causes narcissus commodity value to reduce in various degree.
At present, NLSYV is mainly distributed in the national narcissus cultivation areas such as Britain, Holland, Germany, Italy, Israel, Australia.Because this virus can be carried out long-distance communications with plant and products thereof in spite of illness, thereby strengthen the detection of this virus, to preventing generation and the diffusion of this virus very important.Because NLSYV natural host is narrower, and normal latent infection, utilize conventional biological detection method to identify, not yet find so far effective differential host.Electron microscope observation virion has directly, advantage accurately, but needs expensive plant and instrument and special technician, in the application of port, has larger limitation.ELISA detects has advantages of high specificity, highly sensitive, simple to operate, is current widely used a kind of serology detection technique, and the method weak point is the antiviral antibody that need to have high specificity, good stability.In recent years, develop rapidly along with molecular biological, increasing Protocols in Molecular Biology is applied to the diagnosis qualification of plant virus.Molecular biology for detection based on nucleic acid level, compared with traditional plant method for detecting virus, has the outstanding advantages of short, highly sensitive and high specificity detection time.But up to the present, very few about the report of the slow season yellow of narcissus virus (NLSYV) molecular biology for detection, there is not yet so far the nido RT-PCR technology and the molecular detection kit that are specifically applied to this virus rapid detection.
Summary of the invention
The object of the present invention is to provide a kind of narcissus slow season yellow virus detection kit and detection method thereof, overcome the defect and the deficiency that in prior art, exist, can to pass in and out and agriculture production on the qualification of quarantining fast and accurately of the slow season yellow of narcissus virus.
Technical solution of the present invention is as follows:
(1) detection kit detecting for yellow of slow season of narcissus virus, is characterized in that, described test kit comprises:
1) outside upstream primer: 10 μ mol/L, primer sequence is 5 '-CCACTTGAAGTCTATCACCA-3 ';
2) outside downstream primer: 10 μ mol/L, primer sequence is 5 '-CCAAACTAGCATCCTTGTGT-3 ';
3) inner side upstream primer: 10 μ mol/L, primer sequence is 5 '-ATCACATACACACCACGACA-3 ';
4) inner side downstream primer: 10 μ mol/L, primer sequence is 5 '-GTGTGTCTCTCCGTGTTCTC-3 ';
5）RTBuffer：5×；
6) RNA enzyme inhibition factor: 40U/ μ L;
7) ThermoScript II: 200U/ μ L;
8）dNTPs：10mmol/L；
9）PCR?Buffer：10×；
10）Mgcl
2：25mmol/L；
11) Taq archaeal dna polymerase: 5U/ μ L;
12) positive control sample of the slow season yellow of narcissus virus;
13) not containing the negative control sample of the slow season yellow of narcissus virus;
14）RNase-free?ddH
2O。
(2) detection method of the slow season yellow of above-mentioned narcissus virus detection kit, is characterized in that, comprises the following steps:
1) reverse transcription reaction: adding the total RNA 2 μ L of testing sample, concentration in PCR pipe is outside downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o 5 μ L, 70 DEG C of water-bath 10min, ice bath 5min rapidly, then add following reagent: 5 × RT Buffer, 2.5 μ L, concentration are that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ LRNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, naturally cool to room temperature after 70 DEG C of water-bath 10min, synthetic cDNA;
2) first round PCR reaction: get the synthetic cDNA of step 1) 3 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L outside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH outside 10 μ mol/L
2o 14.5 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 35 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
3) second take turns PCR reaction: get first round PCR reaction product 0.1 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L inside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-freeddH inside 10 μ mol/L
2o 17.4 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
4) pcr amplification product electrophoresis detection: second takes turns PCR reaction finish after, get product 10 μ L 1.5% agarose gel electrophoresis and detect, after ethidium bromide staining, on gel imaging system, observe and record experimental result; The electrophoresis detection result that contains the slow season yellow of narcissus viral sample is to occur bright DNA band at 539bp place.
The present invention is according to yellows virus gene sequences Design Auele Specific Primer of slow season of narcissus, taking the RNA that extracts as the synthetic cDNA of template reverse transcription, utilize cDNA to carry out first round pcr amplification for template, carry out second using first round PCR product as template again and take turns pcr amplification, amplified production carries out agarose gel electrophoresis detection.Compared to prior art, narcissus provided by the present invention slow season yellow virus detection kit and detection method beneficial effect thereof are: 1) high specificity: by using outside, inner side 2 to overlap primer and carry out nest-type PRC, greatly improved the specificity of amplification.Due to the 2nd cover primer (inner side primer) amplified fragments be positioned at the 1st cover primer (outside primer) amplified fragments inside, non-object fragment comprise simultaneously this two cover primer binding site possibility minimum, therefore effectively avoided the generation of non-specific amplification.The present invention can be from infecting the specificity object fragment that the sample amplification of the slow season yellow of narcissus virus is 539bp to size, and the size that all do not increase from other viral sample and healthy sample is the specificity object fragment of 539bp, the invention of result proved has very strong specificity.2) highly sensitive: the present invention takes turns PCR reaction through 2, make detection sensitivity be the order of magnitude and increase, thereby realize the detection to micro-template sample.Compared with conventional RT-PCR, the sensitivity that the present invention detects the slow season yellow of narcissus virus be its 10
2doubly, this has significant application value for carrying the detection few or that do not show disease sample of the slow season yellow of narcissus viral level.3) easy and simple to handle, detection time is short: detection method provided by the invention is easy and simple to handle, detection time is short, can overcome traditional biological mensuration, electron microscopic observation and Serology test and have inefficient deficiency, realize quick, the accurate and efficient detection to the slow season yellow of narcissus virus.
Brief description of the drawings
Fig. 1 is yellow of slow season of the narcissus of embodiment 2 virus detected result.Wherein M:DNA molecular weight standard (100bp); 1: positive control; 2-5: the sample that carries the slow season yellow of narcissus virus; 6: negative control; 7: blank.
Fig. 2 is the specific assay result of embodiment 3.Wherein M:DNA molecular weight standard (100bp); 1: the slow season yellow of narcissus virus (NLSYV) sample; 2: narcissus mosaic virus (NMV) sample; 3: daffod bar virus (NYSV) sample; 4: narcissus degeneration virus (NDV) sample; 5: narcissus cryptovirus (NLV) sample; 6: arabis mosaic virus (ArMV) sample; 7: negative control.
Fig. 3-4 are the sensitivity determination result of embodiment 4 (Fig. 3 is first round PCR measurement result, and Fig. 4 second takes turns PCR measurement result).Wherein M:DNA molecular weight standard (100bp); 1:RNA stoste; 2:10
-1dilution; 3:10
-2dilution; 4:10
-3dilution; 5:10
-4dilution; 6:10
-5dilution; 7:10
-6dilution; 8:10
-7dilution; 9:10
-8dilution; 10:10
-9dilution; 11: negative control; 12: blank.
Embodiment
Below by specific embodiment, the invention will be further described.
Embodiment 1: the configuration (10 detection limits) of the slow season yellow of narcissus virus detection kit
1) outside upstream primer: 10 μ mol/L, 1 pipe (30 μ L);
2) outside downstream primer: 10 μ mol/L, 1 pipe (30 μ L);
3) inner side upstream primer: 10 μ mol/L, 1 pipe (30 μ L);
4) inner side downstream primer: 10 μ mol/L, 1 pipe (30 μ L);
5) RT Buffer:5 ×, 1 pipe (30 μ L);
6) RNA enzyme inhibition factor: 40U/ μ L, 1 pipe (5 μ L);
7) ThermoScript II: 200U/ μ L, 1 pipe (5 μ L);
8) dNTPs:10mmol/L, 1 pipe (30 μ L);
9) PCR Buffer:10 ×, 1 pipe (60 μ L);
10) Mgcl
2: 25mmol/L, 1 pipe (50 μ L);
11) Taq archaeal dna polymerase: 5U/ μ L, 1 pipe (10 μ L);
12) positive control sample of the slow season yellow of narcissus virus, 1 pipe (50 μ L);
13) not containing the negative control sample of the slow season yellow of narcissus virus, 1 manages (50 μ L);
14) RNase-free ddH
2o, 1 pipe (1mL).
Embodiment 2: the detection method of the slow season yellow of narcissus virus detection kit
The detection method of the slow season yellow of above-mentioned narcissus virus detection kit, comprises the following steps:
1) reverse transcription reaction: adding the total RNA 2 μ L of testing sample, concentration in PCR pipe is outside downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o 5 μ L, 70 DEG C of water-bath 10min, rapidly ice bath 5min, then to add following reagent: 5 × RTBuffer 2.5 μ L, concentration be that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ LRNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, naturally cool to room temperature after 70 DEG C of water-bath 10min, synthetic cDNA;
2) first round PCR reaction: get the synthetic cDNA of step 1) 3 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/LMgCl
22 μ L, concentration are that upstream primer 1 μ L outside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH outside 10 μ mol/L
2o 14.5 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 35 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
3) second take turns PCR reaction: get first round PCR reaction product 0.1 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L inside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o 17.4 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
4) pcr amplification product electrophoresis detection: second takes turns PCR reaction finish after, get product 10 μ L 1.5% agarose gel electrophoresis and detect, after ethidium bromide staining, on gel imaging system, observe and record experimental result; The electrophoresis detection result that contains the slow season yellow of narcissus viral sample is to occur bright DNA band (Fig. 1) at 539bp place, otherwise nothing.
Embodiment 3: the specific assay of the slow season yellow of narcissus virus detection kit
1) extraction of the total RNA of narcissus sample: respectively to carry the slow season yellow of narcissus virus (NLSYV), narcissus mosaic virus (Narcissus mosaic virus, NMV), daffod bar virus (Narcissus yellow stripe virus, NYSV), narcissus degeneration virus (NDV), narcissus cryptovirus (Narcissus latent virus, NLV), arabis mosaic virus (Arabis mosaic virus, ArMV) narcissus sample is material, respectively get 0.1g and be placed in mortar, add 1mLPBST damping fluid to grind, 4 DEG C, the centrifugal 5min of 10000g, get supernatant and supernatant liquor be transferred to rapidly in the 1.5mL centrifuge tube of sterilizing, add 1mL TrizoL reagent, after thermal agitation, room temperature leaves standstill 5min, 4 DEG C, the centrifugal 10min of 12000g, gets supernatant, add chloroform 300 μ L, concuss 15s, room temperature leaves standstill 5min, and 4 DEG C, the centrifugal 15min of 12000g, gets upper strata water, add isopyknic Virahol, put upside down and mix standing 15min under rear room temperature, 4 DEG C, the centrifugal 10min of 12000g, abandons supernatant, add the washing with alcohol of 1mL75% to precipitate 2 times, each 4 DEG C, the centrifugal 3min of 7500g, abandons supernatant, after RNA precipitation is dry, with 20 μ L RNase-free ddH
2o dissolves,
2) reverse transcription reaction: adding the total RNA 2 μ L of testing sample, concentration in PCR pipe is outside downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o 5 μ L, 70 DEG C of water-bath 10min, rapidly ice bath 5min, then to add following reagent: 5 × RTBuffer 2.5 μ L, concentration be that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ LRNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, naturally cool to room temperature after 70 DEG C of water-bath 10min, synthetic cDNA;
3) first round PCR reaction: get step 2) synthetic cDNA 3 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L outside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH outside 10 μ mol/L
2o 14.5 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 35 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
4) second take turns PCR reaction: get first round PCR reaction product 0.1 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L inside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o 17.4 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
5) pcr amplification product electrophoresis detection: second takes turns PCR reaction finish after, get product 10 μ L 1.5% agarose gel electrophoresis and detect, after ethidium bromide staining, on gel imaging system, observe and record experimental result (Fig. 2).As seen from Figure 2, there is bright DNA band in the slow season yellow of narcissus viral sample only at 539bp place, and other viral sample and the equal nothing of healthy narcissus sample, illustrate test kit high specificity of the present invention.
Embodiment 4: the sensitivity determination of the slow season yellow of narcissus virus detection kit
The RNA of slow narcissus season yellow virus (NLSYV) sample is diluted to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8with 10
-9doubly, respectively as template, detect by embodiment 2 methods, test arranges negative control and blank simultaneously.From Fig. 3 and Fig. 4, take turns PCR through second, sensitivity can be improved to 10
2doubly, nido RT-PCR can detect and be diluted to 10
-6nLSYV sample RNA doubly, shows that test kit of the present invention has very high sensitivity.
Embodiment 5: the detection of the slow season yellow of narcissus virus on enter the territory narcissus, Chinese narcissus
Choose 8 parts, inward narcissus sample, 72 parts of Chinese narcissus, totally 80 duplicate samples of China's intercept and capture.After extracting the total RNA of all samples, detect by embodiment 2 methods, also adopt IndirectELISA method to detect all samples simultaneously.Result adopts the present invention can from 80 parts of narcissus samples, detect 22 parts, yellow of slow season of narcissus virus, and recall rate is 27.5%, and IndirectELISA only detects 15 parts, and verification and measurement ratio is lower by 8.75% than the present invention.
Claims (2)
1. the slow season yellow of a narcissus virus detection kit, is characterized in that, described test kit comprises: 1) outside upstream primer: 10 μ mol/L, and primer sequence is 5 '-CCACTTGAAGTCTATCACCA-3 '; 2) outside downstream primer: 10 μ mol/L, primer sequence is 5 '-CCAAACTAGCATCCTTGTGT-3 '; 3) inner side upstream primer: 10 μ mol/L, primer sequence is 5 '-ATCACATACACACCACGACA-3 '; 4) inner side downstream primer: 10 μ mol/L, primer sequence is 5 '-GTGTGTCTCTCCGTGTTCTC-3 '; 5) RT Buffer:5 ×; 6) RNA enzyme inhibition factor: 40U/ μ L; 7) ThermoScript II: 200U/ μ L; 8) dNTPs:10mmol/L; 9) PCR Buffer:10 ×; 10) Mgcl
2: 25mmol/L; 11) Taq archaeal dna polymerase: 5U/ μ L; 12) positive control sample of the slow season yellow of narcissus virus; 13) not containing the negative control sample of the slow season yellow of narcissus virus; 14) RNase-freeddH
2o.
2. the detection method of utilizing the slow season yellow of the narcissus virus detection kit described in claim 1, is characterized in that, comprises the following steps:
1) reverse transcription reaction: adding the total RNA 2 μ L of testing sample, concentration in PCR pipe is outside downstream primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o 5 μ L, 70 DEG C of water-bath 10min, rapidly ice bath 5min, then to add following reagent: 5 × RTBuffer 2.5 μ L, concentration be that 10mmol/L dNTPs 1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ LRNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, naturally cool to room temperature after 70 DEG C of water-bath 10min, synthetic cDNA;
2) first round PCR reaction: get the synthetic cDNA of step 1) 3 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L outside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH outside 10 μ mol/L
2o14.5 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 56 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 35 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
3) second take turns PCR reaction: get first round PCR reaction product 0.1 μ L, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs 0.5 μ L, 10 × PCR Buffer, 2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that upstream primer 1 μ L inside 10 μ mol/L, concentration are downstream primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o 17.4 μ L, making to react cumulative volume is 25 μ L; Mixed reaction solution, 94 DEG C of denaturation 3min, then 94 DEG C of sex change 30s, 58 DEG C of annealing 45s, 72 DEG C of extension 1min, totally 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
4) pcr amplification product electrophoresis detection: second takes turns PCR reaction finish after, get product 10 μ L 1.5% agarose gel electrophoresis and detect, after ethidium bromide staining, on gel imaging system, observe and record experimental result; The electrophoresis detection result that contains the slow season yellow of narcissus viral sample is to occur bright DNA band at 539bp place.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310416761.0A CN103484567B (en) | 2013-09-13 | 2013-09-13 | Narcissus late season yellows virus detection kit and method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310416761.0A CN103484567B (en) | 2013-09-13 | 2013-09-13 | Narcissus late season yellows virus detection kit and method |
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| Publication Number | Publication Date |
|---|---|
| CN103484567A CN103484567A (en) | 2014-01-01 |
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| CN102690896A (en) * | 2012-05-16 | 2012-09-26 | 福建出入境检验检疫局检验检疫技术中心 | Kit for detecting narcissus mosaic virus and narcissus yellow stripe virus and detection method of kit |
| CN102732642A (en) * | 2012-05-14 | 2012-10-17 | 福建出入境检验检疫局检验检疫技术中心 | Narcissus latent virus detection kit and detection method thereof |
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| CN102732642A (en) * | 2012-05-14 | 2012-10-17 | 福建出入境检验检疫局检验检疫技术中心 | Narcissus latent virus detection kit and detection method thereof |
| CN102690896A (en) * | 2012-05-16 | 2012-09-26 | 福建出入境检验检疫局检验检疫技术中心 | Kit for detecting narcissus mosaic virus and narcissus yellow stripe virus and detection method of kit |
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