CN104120192A - Nest type RT-PCR detection kit and detection method for narcissus degeneration virus - Google Patents
Nest type RT-PCR detection kit and detection method for narcissus degeneration virus Download PDFInfo
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Abstract
The invention relates to a nest type RT-PCR detection kit and a detection method for narcissus degeneration virus, which are specially used for detecting narcissus degeneration virus. The kit comprises an outer-side forward primer, an outer-side reverse primer, an inner-side forward primer, an inner-side reverse primer, an RT Buffer, an RNA enzyme inhibiting factor, a reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, Taq DNA polymerase, a positive control, a negative control and RNase-free ddH2O. The nest type RT-PCR detection kit and the detection method conduct detection to the narcissus degeneration virus by adopting nest type RT-PCR technology, and have the advantages of being strong in specificity, high in sensitivity, simple and convenient to operate, and fast in detection, and are applicable to fast detection, epidemic situation monitoring and early-warning and forecasting on the narcissus degeneration virus in China export and import quarantine and agricultural production, thus being wide in application prospects.
Description
Technical field
The present invention relates to a kind of narcissus degenerate viral nido RT-PCR detection kit and detection method thereof, belong to Plant Quarantine technical field, be applicable to pass in and out and agriculture production on narcissus degenerate viral rapid detection and monitoring.
Background technology
Narcissus degeneration virus (Narcissus degeneration virus, NDV) be marmor upsilon section (Potyviridae) Potyvirus (Potyvirus) member, viral genome total length approximately 9,816bp, for sense single stranded rna,, containing polyA tail, this virus does not produce typical pinwheel inclusion in the narcissus mesophyll cell being infected.This virus, as far back as the upper discovery of Britain's polyanthus mutation (Narcissus tazetta cv.Grand Soleil d ' Or), shows as chlorisis striped symptom on the narcissus blade being infected.NDV experiment host is narrower, mainly be confined on the several plant of Amaryllidaceae (Amaryllidaceae), plant that can frictional inoculation comprises Chinese narcissus (Narcissus tazetta var.chinensis Roem), narcissus poeticus (Narcissus poeticus), three kinds of narcissuses of daffadowndilly (Narcissus pseudonarcissus) and short-tube lycoris (Lycoris radiata) plant.The genom sequence size of NDV is 9816 Nucleotide, there is the typical genome structure of potyvirus, containing a large open reading frame (ORF), the polyprotein of 3184 amino-acid residues of coding, molecular weight is 363.4KDa.NDV virion is filament shape, mainly propagates between its plant via aphid vector.NDV on serological relation with marmor upsilon (Potato virus Y, PVY), Brassica 2 et 4 (Turnip mosaic virus, TuMV) there is serology dependency, but there is no serological relation with another slow flavivirus of narcissus (Narcissus late season yellows virus, NLSYV) that is all potyvirus.In addition, NDV can also with other plant virus Combined Infection narcissus, affect the quality of narcissus, cause narcissus commodity value to determine to reduce in degree in difference.
At present, NDV is mainly distributed in the narcissus cultivation areas such as Britain, Germany, New Zealand, Australia, China.The long-distance communications of NDV mainly rely on the transport of carrying this viral plant and products thereof, therefore strengthen the detection of this virus, are conducive to prevent generation and the diffusion of this virus.Because NDV host range is narrow, and often there is Combined Infection, utilize traditional biological detection method to detect and not yet find effective differential host; Utilize Electronic Speculum detection method to observe virion and there is directly and accurately advantage, but it needs special technician and expensive plant and instrument, makes it in the application at port, have larger limitation; ELISA detection method is highly sensitive, high specificity, easy and simple to handle, be a kind of serology detection technique of current widespread use, but its limitation is to need the antiviral antibody of good stability, high specificity.In recent years, round pcr is widely used in the detection of plant virus, highly sensitive, high specificity.Nido RT-PCR method based on conventional RT-PCR, can reduce the possibility that multiple targets are amplified, and reduces false-positive appearance, improves the sensitivity and the reliability that detect, has outstanding advantages.But up to the present, very few about the report of narcissus degeneration virus (NDV) nido RT-PCR detection method, there is not yet so far the nido RT-PCR detection kit that is specifically applied to this virus rapid detection.
Summary of the invention
The object of the present invention is to provide a kind of narcissus degenerate viral nido RT-PCR 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 narcissus degeneration virus.
Technical solution of the present invention is as follows:
(1) the nido RT-PCR detection kit detecting for narcissus degeneration virus, is characterized in that, described test kit comprises:
1) outside forward primer: 10 μ mol/L, primer sequence is 5 '-ACCGTTGGCATCACTAAG-3 ';
2) outside reverse primer: 10 μ mol/L, primer sequence is 5 '-ACTGGTTCCACGCTCCTA-3 ';
3) inner side forward primer: 10 μ mol/L, primer sequence is 5 '-GCGGTGATAACAATTCGAGA-3 ';
4) inner side reverse primer: 10 μ mol/L, primer sequence is 5 '-GCTAAGTCCTTCAATTTCCGTC-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) the narcissus viral positive control sample of degenerating;
13) not containing the negative control sample of narcissus Degenerative disease poison;
14)RNase-free ddH
2O。
(2) the above-mentioned narcissus detection method of viral nido RT-PCR detection kit of degenerating, is characterized in that, comprises the following steps:
1) reverse transcription reaction: adding the total RNA3 μ of testing sample L, concentration in PCR pipe is outside reverse primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o7 μ L, 70 DEG C of water-bath 10min, ice bath 5min rapidly, then add following reagent: 5 × RT Buffer2.5 μ L, concentration are that 10mmol/L dNTPs1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, 70 DEG C of water-bath 10min, synthetic cDNA;
2) the 1st take turns PCR reaction: get step 1) synthetic cDNA3 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 dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L outside 10 μ mol/L, concentration are reverse 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 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
3) the 2nd take turns PCR reaction: get the first round PCR reaction product after 0.2 μ L dilutes 100 times, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L inside 10 μ mol/L, concentration are reverse primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o17.3 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: the 2nd 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 narcissus degeneration viral sample is to occur bright DNA band at 393bp place.
According to the narcissus Degenerative disease virus gene sequences Design two of having reported to Auele Specific Primer, to extract RNA as the synthetic cDNA of template reverse transcription is as template, carry out first round pcr amplification, carry out second using the product of first round pcr amplification as template again and take turns pcr amplification, its amplified production carries out agarose gel electrophoresis detection.Compared with prior art, degenerate viral nido RT-PCR detection kit and detection method beneficial effect thereof of narcissus provided by the present invention is: 1) high specificity: by using two pairs, interior outside primer to carry out nest-type PRC, greatly improved the specificity of viral detection.The amplification site of inner side primer (second pair of primer) is positioned at the inside, amplification site of outside primer (pair of primers), it is minimum that non-object fragment comprises the possibility of this two covers primer binding site simultaneously, therefore effectively avoided the appearance in multiple targets site.The specificity object fragment that the sample amplification that the present invention can degenerate viral from infection narcissus is 393bp to size, and the size that all do not increase from healthy sample and other viral sample is the specificity object fragment of 393bp, 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.The present invention is with respect to regular-PCR technology, detect narcissus degenerate viral sensitivity be its 10
2-10
3doubly, there is significant application value for not showing disease sample or carrying the viral poor detection of narcissus degeneration.3) easy and simple to handle, the cycle is short: detection method provided by the invention is easy and simple to handle, the cycle is short, overcome traditional biological mensuration, electron microscopic observation and Serology test and have the not high shortcoming of sensitivity, realize quick, the accurate and efficient detection to narcissus degeneration virus.
Brief description of the drawings
Fig. 1 is the narcissus of the embodiment 2 viral detected result of degenerating.Wherein M:DNA molecular weight standard (100bp); 1: positive control; 2: negative control; 3: blank; 4-5: carry the narcissus viral sample of degenerating; 6-7: do not carry the narcissus viral sample of degenerating.
Fig. 2 is the specific assay result of embodiment 3.Wherein M:DNA molecular weight standard (100bp); 1: narcissus degeneration virus (NDV) sample; 2: narcissus mosaic virus (NMV) sample; 3: daffod bar virus (NYSV) sample; 4: the slow season yellow of narcissus virus (NLSYV) sample; 5: narcissus cryptovirus (NLV) sample; 6: arabis mosaic virus (ArMV) sample; 7: negative control.
Fig. 3 is that the 1st of embodiment 4 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; 11:10
-10; 12: negative control.
Fig. 4 is that the 2nd of embodiment 4 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; 11:10
-10; 12: negative control.
Embodiment
Below by specific embodiment, the invention will be further described.
Embodiment 1: the degenerate configuration (10 detection limits) of viral nido RT-PCR detection kit of narcissus
1) outside forward primer: 10 μ mol/L, 1 pipe (30 μ L);
2) outside reverse primer: 10 μ mol/L, 1 pipe (30 μ L);
3) inner side forward primer: 10 μ mol/L, 1 pipe (30 μ L);
4) inner side reverse 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) the narcissus viral positive control sample of degenerating, 1 pipe (50 μ L);
13) not containing the negative control sample of narcissus Degenerative disease poison, 1 manages (50 μ L);
14) RNase-free ddH
2o, 1 pipe (1mL).
Embodiment 2: the degenerate detection method of viral nido RT-PCR detection kit of narcissus
The degenerate detection method of viral nido RT-PCR detection kit of above-mentioned narcissus, comprises the following steps:
1) reverse transcription reaction: adding the total RNA3 μ of testing sample L, concentration in PCR pipe is outside reverse primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o7 μ L, 70 DEG C of water-bath 10min, ice bath 5min rapidly, then add following reagent: 5 × RT Buffer2.5 μ L, concentration are that 10mmol/L dNTPs1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, 70 DEG C of water-bath 10min, synthetic cDNA;
2) the 1st take turns PCR reaction: get step 1) synthetic cDNA3 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 dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L outside 10 μ mol/L, concentration are reverse 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 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
3) the 2nd take turns PCR reaction: get the first round PCR reaction product after 0.2 μ L dilutes 100 times, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L inside 10 μ mol/L, concentration are reverse primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o17.3 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: the 2nd 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 narcissus degeneration viral sample is to occur bright DNA band (Fig. 1) at 393bp place, otherwise nothing.
Embodiment 3: the degenerate specific assay of viral nido RT-PCR detection kit of narcissus
1) extraction of the total RNA of narcissus sample: respectively to carry narcissus degeneration virus (NDV), the slow season yellow of narcissus virus (NLSYV), narcissus mosaic virus (Narcissus mosaic virus, NMV), daffod bar virus (Narcissus yellow stripe virus, NYSV), 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 1mL PBST 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 RNA3 μ of testing sample L, concentration in PCR pipe is outside reverse primer 1 μ L and the RNase-free ddH2O7 μ L of 10 μ mol/L, 70 DEG C of water-bath 10min, ice bath 5min rapidly, then add following reagent: 5 × RT Buffer2.5 μ L, concentration are that 10mmol/L dNTPs1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, 70 DEG C of water-bath 10min, synthetic cDNA;
3) the 1st take turns PCR reaction: get step 1) synthetic cDNA3 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 dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L outside 10 μ mol/L, concentration are reverse 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 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
4) the 2nd take turns PCR reaction: get the first round PCR reaction product after 0.2 μ L dilutes 100 times, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L inside 10 μ mol/L, concentration are reverse primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o17.3 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: the 2nd 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 narcissus degeneration viral sample only at 393bp place, and other viral sample and the equal nothing of healthy narcissus sample, illustrate that test kit of the present invention has very strong specificity.
Embodiment 4: the degenerate sensitivity determination of viral nido RT-PCR detection kit of narcissus
The RNA of narcissus degeneration virus (NDV) 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 the 2nd, sensitivity can be improved to 10
4doubly, nido RT-PCR can detect and be diluted to 10
-9nDV sample RNA doubly, shows that test kit of the present invention has very high sensitivity.
Embodiment 5: the narcissus viral detection of degenerating on enter the territory narcissus, Chinese narcissus
Choose 15 parts, the narcissus sample of China's intercept and capture, detect by embodiment 2 methods after extracting the total RNA of all samples.Result adopts the present invention can from 11 parts of narcissus samples, detect narcissus degeneration virus, and recall rate reaches 73.3%.
<110> Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
<120> narcissus degenerate viral nido RT-PCR detection kit and detection method thereof
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
accgttggca tcactaag 18
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
actggttcca cgctccta 18
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
gcggtgataa caattcgaga 20
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
gctaagtcct tcaatttccg tc 22
Claims (2)
1. the narcissus viral nido RT-PCR detection kit of degenerating, is characterized in that, described test kit comprises: 1) outside forward primer: 10 μ mol/L, and primer sequence is 5 '-ACCGTTGGCATCACTAAG-3 '; 2) outside reverse primer: 10 μ mol/L, primer sequence is 5 '-ACTGGTTCCACGCTCCTA-3 '; 3) inner side forward primer: 10 μ mol/L, primer sequence is 5 '-GCGGTGATAACAATTCGAGA-3 '; 4) inner side reverse primer: 10 μ mol/L, primer sequence is 5 '-GCTAAGTCCTTCAATTTCCGTC-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) Mgcl2:25mmol/L; 11) Taq archaeal dna polymerase: 5U/ μ L; 12) the narcissus viral positive control sample of degenerating; 13) not containing the negative control sample of narcissus Degenerative disease poison; 14) RNase-free ddH
2o.
2. utilize the degenerate detection method of viral nido RT-PCR detection kit of narcissus described in claim 1, it is characterized in that, comprise the following steps:
1) reverse transcription reaction: adding the total RNA3 μ of testing sample L, concentration in PCR pipe is outside reverse primer 1 μ L and the RNase-free ddH of 10 μ mol/L
2o7 μ L, 70 DEG C of water-bath 10min, ice bath 5min rapidly, then add following reagent: 5 × RT Buffer2.5 μ L, concentration are that 10mmol/L dNTPs1 μ L, concentration are that 200U/ μ L ThermoScript II 0.5 μ L, concentration are 40U/ μ L RNA enzyme inhibition factor 0.5 μ L.42 DEG C of water-bath 60min, 70 DEG C of water-bath 10min, synthetic cDNA;
2) the 1st take turns PCR reaction: get step 1) synthetic cDNA3 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 dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L outside 10 μ mol/L, concentration are reverse 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 30 circulations so, after last loop ends, 72 DEG C are continued to extend 10min, and reaction finishes;
3) the 2nd take turns PCR reaction: get the first round PCR reaction product after 0.2 μ L dilutes 100 times, adding concentration by every pipe is that 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, concentration are that 10mmol/L dNTPs0.5 μ L, 10 × PCR Buffer2.5 μ L, concentration are 25mmol/L MgCl
22 μ L, concentration are that forward primer 1 μ L inside 10 μ mol/L, concentration are reverse primer 1 μ L, RNase-free ddH inside 10 μ mol/L
2o17.3 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: the 2nd 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 narcissus degeneration viral sample is to occur bright DNA band at 393bp place.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878123A (en) * | 2015-05-12 | 2015-09-02 | 福建省农业科学院果树研究所 | Blueberry shock virus nested RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method thereof |
| CN105525037A (en) * | 2015-07-24 | 2016-04-27 | 湖南农业大学 | Nested RT-PCR method and primer combination for detecting potato mop-top virus |
| CN108559793A (en) * | 2018-06-19 | 2018-09-21 | 福建省农业科学院果树研究所 | A kind of the TC-RT-nested-PCR detection kits and its detection method of cordate telosma mosaic virus |
| CN109536642A (en) * | 2018-12-19 | 2019-03-29 | 长江大学 | A kind of universal pig fourth type coronavirus RT-Nested PCR detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2014
- 2014-06-26 CN CN201410294845.6A patent/CN104120192B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| CHEN,J. ET AL.: "Accession No: AM182028，Narcissus degeneration virus complete genome, isolate Zhangzhou", 《GENEBANK DATABASE》 * |
| 顾青雷等: "PCR 相关技术在植物病毒检疫检测中的应用", 《生物技术通报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878123A (en) * | 2015-05-12 | 2015-09-02 | 福建省农业科学院果树研究所 | Blueberry shock virus nested RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method thereof |
| CN105525037A (en) * | 2015-07-24 | 2016-04-27 | 湖南农业大学 | Nested RT-PCR method and primer combination for detecting potato mop-top virus |
| CN108559793A (en) * | 2018-06-19 | 2018-09-21 | 福建省农业科学院果树研究所 | A kind of the TC-RT-nested-PCR detection kits and its detection method of cordate telosma mosaic virus |
| CN109536642A (en) * | 2018-12-19 | 2019-03-29 | 长江大学 | A kind of universal pig fourth type coronavirus RT-Nested PCR detection method |
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