CN101818211A - Molecule detection method for cowpea severe mosaic virus - Google Patents
Molecule detection method for cowpea severe mosaic virus Download PDFInfo
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Abstract
The invention relates to a molecule detection method for cowpea severe mosaic virus, which belongs to the technical field of biology. The method comprises the following steps of: obtaining coarse virus extract; preparing an immune capturing PCR tube; releasing cowpea severe mosaic virus RNA in the immune capturing PCR tube and taking the cowpea severe mosaic virus RNA as a template, and performing reverse transcription reaction by using CPSMVR of a sequence 5'-GGCTTCTGCAGGTGTTCCAA-3' as a primer to obtain a reverse transcription product; and performing real-time fluorescent PCR reaction by using the reverse transcription product as a template and using a forward primer 5'-GGTCAATCCCGGCATTATTG-3', a reverse primer CPSMVR and a Taq Man probe CPSMVPro 5'FAM-TGTAGCACAATCAGGGCAAACACAGCA-TAMRA 3', wherein when the initial cycle number is less than or equal to 35, a sample is infected with the cowpea severe mosaic virus.
Description
Technical field
The present invention relates to a kind of detection method of cowpea severe mosaic virus, particularly immunocapture-reverse transcription-real-time fluorescence PCR molecular detecting method (or claiming IC-RT-Real time PCR method) belongs to biological technical field.
Background technology
(Cowpea severe mosaic virus CPSMV) belongs to definite kind of Comoviridae (Comoviridae) Comovirus (Comovirus) to cowpea severe mosaic virus.This viral natural host is a leguminous plants, can seriously infect various crop such as cowpea, soybean.After this virus infection cowpea, can cause blade floral leaf and mottled, serious causes whole strain withered; Infection rate in the field to cowpea can reach 100%, causes production loss can reach 50%.
CPSMV mainly is distributed in the area, America at present, and as states such as the U.S., Brazil, Peru, Venezuela, Trinida, Puerto Rico, Costa Rica and Suriname, China does not still have the report that takes place with harm.
CPSMV can carry out long-distance communications by seed, and wherein the kind biography rate of cowpea is 10%, and the kind biography rate of asparagus bean (Vignasesquipedalis) is 8%; In the field, this virus can also be passed through Kidney bean chrysomelid (Ceratomatrifurcata), band spot cucumber chrysomelid (Diabrotica balteata) and South America chrysomelid insect vector and mechanical inoculations such as (D.speciosa).This viral natural host is extensively planted in China, and weather condition are similar to the weather condition of this virus spot, and therefore, the possibility that CPSMV imports into, grows surely and spread is all very big, and can propagate at home with the allocation and transportation and the insect of seed.CPSMV has become China leguminous plants especially cowpea and soybean production potential grave danger.
In view of this viral natural host cowpea and soybean etc. are the main cash crop of China, can cause very big harm to it, China lists this virus in " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register ".
At present, the quarantine port utilizes traditional DAS-ELISA (double antibodies sandwich enzyme-linked immunosorbent assay) technology to detect CPSMV more, and is quick inadequately, sensitive, accurate.
Summary of the invention
The purpose of this invention is to provide the cowpea severe mosaic virus IC-RT-Real time PCR molecular detecting method that use at a kind of port that is suitable for quarantining so that fast, sensitive, the cowpea seed of port import is carried out the detection of cowpea severe mosaic virus exactly.
The invention provides a kind of molecule detection method for cowpea severe mosaic virus, concrete steps are as follows:
(1) preparation of sample virus crude extract: in sample to be checked, add extraction buffer,, get supernatant liquor, be viral crude extract through grinding and centrifugal;
(2) preparation of immunocapture PCR pipe: get cowpea severe mosaic virus coated antibody after the dilution and add in the PCR pipe and hatch, after the lavation buffer solution washing, add the described viral crude extract of step 1,4 ℃ of refrigerator overnight incubation, the washing back is air-dry, promptly obtains immunocapture PCR pipe;
(3) cowpea severe mosaic virus RNA discharges and reverse transcription in the immunocapture PCR pipe: the cowpea severe mosaic virus RNA in the described immunocapture PCR of release steps (2) pipe and be template with it, with sequence be: the CPSMVR of 5 '-GGCTTCTGCAGGTGTTCCAA-3 ' is that primer carries out reverse transcription reaction, obtains reverse transcription product;
(4) real-time fluorescence PCR reaction: with step (3) gained reverse transcription product is template, forward primer CPSMVF sequence: 5 '-GGTCAATCCCGGCATTATTG-3 ', the sequence of reverse primer CPSMVR: 5 '-GGCTTCTGCAGGTGTTCCAA-3 ', the sequence of Taq Man probe CPSMVPro: 5 ' FAM-TGTAGCACAATCAGGGCAAACACAGCA-TAMRA 3 ', carry out the real-time fluorescence PCR reaction;
(5) interpretation of result: after the described real-time fluorescence PCR reaction of step 4 finishes, utilize real-time fluorescence PCR instrument analysis software, the collection of illustrative plates of amplification is analyzed, as initial cycle number (C
T)≤35 o'clock judge that the real-time fluorescence PCR reaction is positive, and the viral sample that expression is detected is a cowpea severe mosaic virus.
The detailed process of cowpea severe mosaic virus RNA release and reverse transcription is in the described immunocapture PCR of step (3) pipe: add viral RNA and discharge liquid in described immunocapture PCR pipe, handle 5min for 95 ℃ then, rapidly after the cooling, add following material again and carry out reverse transcription: 5 * Buffer of 2 μ L, 0.5 the RT Enzyme MixI of μ L, response procedures is: 42 ℃ of reaction 60min; The concentration that consists of 0.5 μ L that described viral RNA discharges liquid is the ddH of the no RNA enzyme of the reverse primer CPSMVR of 10 μ mol/L and 7.0 μ L
2O.
The described real-time fluorescence PCR reaction system of step 4 contains: 2 * Premix Ex Taq of 10 μ L, and concentration is CPSMVF and each 0.8 μ L of CPSMVR of 10 μ mol/L, 0.2 μ L concentration is the CPSMVPro of 10 μ mol/L, 2 μ L reverse transcription products, the ddH of 6.2 μ L
2O supplies volume to 20 μ L; The reaction conditions of described real-time fluorescence PCR: 94 ℃ of reaction 3min; 94 ℃ of reaction 15sec, 60 ℃ of reactions 60sec, totally 45 circulations.
Immunocapture-reverse transcription-the real-time fluorescence PCR detection method of the cowpea severe mosaic virus that the present invention sets up, combine the advantage of antigen and antibody specific combination, molecular hybridization, three kinds of technology of Real time-PCR (real-time fluorescence PCR), compare with traditional RT-PCR (reverse transcription PCR) method, DAS-ELISA (double antibodies sandwich enzyme-linked immunosorbent assay) method and molecular hybridization, have advantages such as specificity is good, highly sensitive, good reproducibility.Can directly detect cowpea seed or plant by the characteristics of seed dispersal at this virus simultaneously.This method has been saved the RNA extractive process, has avoided that polysaccharide and some aldehydes matters exert an influence to extracting viral RNA and reverse transcription reaction in the plant host, can prevent false positive, false-negative generation preferably.The present invention is fit to the detection to cowpea severe mosaic virus in legume vegetable seed such as the cowpea of passing in and out and the seedling, especially is fit to the detection of virus concentration when low, has a good application prospect.
Description of drawings
Fig. 1 is cowpea severe mosaic virus (CPSMV) IC-RT-Real time pcr amplification curve (CPSMV-infects the sample of cowpea severe mosaic virus, and CK-represents negative control)
Embodiment
Concrete detection method is as follows:
1. the preparation of sample virus crude extract
1.1 infect selecting and sowing of CPSMV seed: infect symptom feature after this virus according to cowpea, it is little to select grain, the seed of symptoms such as grain is flat, behind 3% (m/v) clorox surface sterilization 10min, with sterilized water washing three times, in the soil of sterilizing, employed basin alms bowl and utensil are all through disinfecting with this planting seed.
1.2 infected plant selects and the preparation of viral crude extract: after cowpea is emerged and is flattened to first pair of true leaf, to performance suspect plant with sample of individual plant preparation; For asymptomatic plant, 1 leaf is got in every strain, sample of per 10 strain blades preparation.Fresh leaf tissue in each sample has 0.1g, grinds to wherein adding sample extraction buffer 1mL, and centrifugal 10min under centrifugal force 10000g condition, supernatant liquor is viral crude extract.Also the seed after the surface sterilization directly can be added the sample extraction buffer, grind and centrifugal after make viral crude extract.
Wherein the composition of extraction buffer is as follows: 2% (mass concentration) Polyvinylpyrolidone (PVP), 0.05% (mass concentration) tween 20, pH7.4 phosphate buffered saline buffer (0.01mol/L)
2. the preparation of immunocapture PCR pipe
Get the CPSMV coated antibody, being cushioned liquid with bag is to dilute at 1: 200 by volume, and the CPSMV coated antibody of drawing after the 25 μ L dilution adds in the PCR pipe, and room temperature is placed 4h or 4 ℃ of refrigerator overnight incubation; After washing 3 times with lavation buffer solution (PBST), add 25 μ L virus crude extract, after 4 ℃ of refrigerator overnight incubation, wash 3 times, sterilization distilled water (ddH with PBST
2O) wash 2 times, air-dry 30min promptly obtains immunocapture PCR pipe.Can carry out reverse transcription or put-20 ℃ refrigerator prolonged preservation standby thereafter.
Wherein the CPSMV coated antibody derives from cowpea severe mosaic virus DAS-ELISA detection kit, German DSMZ company product; Bag is cushioned the liquid composition: 15mmol/L yellow soda ash, 35mmol/L sodium bicarbonate, pH9.6.
3. cowpea severe mosaic virus RNA discharges and reverse transcription in the immunocapture PCR pipe
Add viral RNA and discharge liquid in the immunocapture PCR pipe that has prepared: the concentration of 0.5 μ L is the ddH of the no RNA enzyme of the reverse primer CPSMVR of 10 μ mol/L and 7.0 μ L
2O handles 5min for 95 ℃ on the PCR instrument, after the cooling, add residue reverse transcription reaction liquid system again and carry out reverse transcription rapidly.
Residue reverse transcription reaction liquid system:
5×Buffer(for?Real?Time)：2μL
RT?Enzyme?Mix?I：0.5μL
Reverse transcription reaction condition: 42 ℃ of reaction 60min.
The reagent source that this reverse transcription reaction is used is in PrimeScript
TMRT-PCR test kit (Perfect RealTime), precious biotechnology (Dalian) company limited product.
4. real-time fluorescence PCR reaction
4.1CPSMV Taq Man probe and primer design and synthetic
(accession number: the conserved sequence NC003544) designs a pair of Oligonucleolide primers according to CPSMV coat protein encoding gene, RNA (the standard bacterium numbering PV-273 of American Type Culture Collecti) with CPSMV is that template checks order after RT-PCR amplifies product, according to sequencing result, design following a pair of Oligonucleolide primers and Taq Man probe:
CPSMVF (forward primer): 5 '-GGTCAATCCCGGCATTATTG-3 '
CPSMVR (reverse primer): 5 '-GGCTTCTGCAGGTGTTCCAA-3 '
CPSMVPro (Taq Man probe): 5 ' (FAM)-TGTAGCACAATCAGGGCAAACACAGCA-(TAMRA) 3 ' (FAM is the fluorescence report group, and TAMRA is the fluorescent quenching group)
The amplified production size of its expection is 109bp; Above Taq Man probe and PCR primer are synthetic by precious biotechnology (Dalian) company limited.
4.2 real-time fluorescence PCR reaction
The explanation of reference reagent box, prepare following component real-time fluorescence PCR reaction system (20 μ L):
2×Premix?Ex?Taq：10μL
CPSMVF(10μmol/L)：0.8μL
CPSMVR(10μmol/L)：0.8μL
CPSMVPro(10μmol/L)：0.2μL
Template (reverse transcription product): 2 μ L
ddH
2O：6.2μL
Real time-PCR reaction conditions:
94 ℃ of reaction 3min; 94 ℃ of reaction 15sec, 60 ℃ of reaction 60sec, 45 circulations.
Reagent source in the real-time fluorescence PCR reaction is in PrimeScript
TMRT-PCR test kit (Perfect RealTime), precious biotechnology (Dalian) company limited product.
5. interpretation of result
The real-time fluorescence PCR amplified reaction utilizes real-time fluorescence PCR instrument analysis software after finishing, and the collection of illustrative plates of amplification is analyzed, as initial cycle number (C
T)≤35 o'clock judge that the real-time fluorescence PCR reaction is positive, and the viral sample that expression is detected is cowpea severe mosaic virus (as Fig. 1).
The inventive method of utilizing embodiment 2 detects the specificity of cowpea severe mosaic virus
1. the viral RNA that is used for the specificity checking extracts
Choose the cowpea mosaic virus (CPMV) and the bean pod mottle virus (BPMV) that belong to (Comovirus Comovirus) with cowpea severe mosaic virus together, another one Nepovirus (Nepovirus) nepovirus (TRSV) of equal (Comoviridae Comoviridae), and the black eye cowpea mosaic virus (BlCMV) that the important kind of another one passes viral Potyvirus on the cowpea, carry out the checking of cowpea severe mosaic virus (CPSMV) RT-Real time PCR specificity.The virus of more than participating in the experiment is U.S. representative microbial DSMZ (American Type Culture Collection is called for short ATCC).Negative control is the blade of healthy cowpea.
In infecting each viral plant leaf (0.1g), add sample extraction buffer 1mL respectively and grind, centrifugal 10min under centrifugal force 10000g condition, supernatant liquor is viral crude extract.
Wherein the composition of extraction buffer is as follows: 2% (mass concentration) Polyvinylpyrolidone (PVP), 0.05% (mass concentration) tween 20, pH7.4 phosphate buffered saline buffer (0.01mol/L)
2. the preparation of immunocapture PCR pipe
Get the CPSMV coated antibody, being cushioned liquid with bag is to dilute at 1: 200 by volume, and the CPSMV coated antibody of drawing after the 25 μ L dilution adds in the PCR pipe, and room temperature is placed 4h or 4 ℃ of refrigerator overnight incubation; After washing 3 times with lavation buffer solution (PBST), add 25 μ L virus crude extract, after 4 ℃ of refrigerator overnight incubation, wash 3 times, sterilization distilled water (ddH with PBST
2O) wash 2 times, air-dry 30min promptly obtains immunocapture PCR pipe.Can carry out reverse transcription or put-20 ℃ refrigerator prolonged preservation standby thereafter.
Wherein the CPSMV coated antibody derives from cowpea severe mosaic virus DAS-ELISA detection kit, German DSMZ company product; Bag is cushioned the liquid composition: 15mmol/L yellow soda ash, 35mmol/L sodium bicarbonate, pH9.6.
3. cowpea severe mosaic virus RNA discharges and reverse transcription in the immunocapture PCR pipe
Add viral RNA and discharge liquid in the immunocapture PCR pipe that has prepared: the concentration of 0.5 μ L is the ddH of the no RNA enzyme of the reverse primer CPSMVR of 10 μ mol/L and 7.0 μ L
2O handles 5min for 95 ℃ on the PCR instrument, after the cooling, add the residue inverse transcription reaction liquid again and carry out reverse transcription rapidly.
Residue reverse transcription reaction liquid system:
5×Buffer(for?Real?Time)：2μL
RT?Enzyme?Mix?I：0.5μL
Reverse transcription reaction condition: 42 ℃ of reaction 60min.
The reagent source that this reverse transcription reaction is used is in PrimeScript
TMRT-PCR test kit (Perfect RealTime), precious biotechnology (Dalian) company limited product.
4. real-time fluorescence PCR reaction
Prepare following component real-time fluorescence PCR reaction system (20 μ L):
2×Premix?Ex?Taq：10μL
CPSMVF(10μmol/L)：0.8μL
CPSMVR(10μmol/L)：0.8μL
CPSMVPro(10μmol/L)：0.2μL
Template (reverse transcription product): 2 μ L
ddH
2O：6.2μL
Real time-PCR reaction conditions:
94 ℃ of reaction 3min; 94 ℃ of reaction 15sec, 60 ℃ of reaction 60sec, 45 circulations.
4. interpretation of result
Have only in the real-time fluorescence PCR reaction of CPSMV, amplification curve (C is arranged
TValue is 12.12), and other four viruses all do not have amplification, show that designed cowpea severe mosaic virus primer and probe can satisfy the requirement of Real time PCR specific reaction.
The inventive method of utilizing embodiment 3 detects the sensitivity of cowpea severe mosaic virus IC-RT-Real time PCR
1. the preparation of cowpea severe mosaic virus sample crude extract
Take by weighing the sick 100mg of leaf texture of cowpea severe mosaic virus, add sample extraction buffer 1mL and grind, the centrifugal 10min of 10000g, supernatant liquor is viral crude extract, and the concentration of supernatant liquor is 100mg/mL virus crude extract.Be respectively the viral crude extract of 10mg/mL, 1mg/mL, 100 μ g/mL, 10 μ g/mL, 10ng/mL and 1ng/mL again with 10 times of gradient stepwise dilution to concentration of sample extraction buffer.
2. the preparation of immunocapture PCR pipe
Get the CPSMV coated antibody, after being cushioned liquid and being diluted to working concentration with bag, draw 25 μ L and add in the PCR pipe, room temperature is placed 4h or 4 ℃ of refrigerator overnight incubation.Wash 3 times with lavation buffer solution (PBST), get 25 μ L virus coarse body fluid and add in the coated antibody PCR pipe, 4 ℃ of refrigerator overnight incubation are washed 3 times with PBST, sterilization distilled water (ddH
2O) wash 2 times, air-dry 30min carries out reverse transcription or puts-20 ℃ of refrigerator prolonged preservation standby.
3. viral RNA discharges and reverse transcription in the immunocapture PCR pipe
Add viral RNA and discharge liquid in the immunocapture PCR pipe that has prepared: 0.5 μ L, concentration are the CPSMVR of 10 μ mol/L and the ddH that does not have the RNA enzyme
2O 7.0 μ L handle 5min for 95 ℃ on the PCR instrument, after the cooling, add the residue inverse transcription reaction liquid again and carry out reverse transcription rapidly.
Residue RT reaction solution system:
5×Buffer(for?Real?Time)：2μL
RT?Enzyme?Mix?I：0.5μL
Reverse transcription reaction condition: 42 ℃ of reaction 60min
4. real-time fluorescence PCR reaction
Press following set of dispense system real-time fluorescence PCR reaction system (20 μ L):
Premix?Ex?Taq(2×)：10μL
CPSMVF(10μmol/L)：0.8μL
CPSMVR(10μmol/L)：0.8μL
CPSMVPro(10μmol/L)：0.2μL
Template (reverse transcription product): 2 μ L
ddH
2O：6.2μL
The reaction conditions of real-time fluorescence PCR:
94 ℃ of 3min; 94 ℃, 15sec; 60 ℃, 60sec; Totally 45 circulations.
5. interpretation of result
After immunocapture PCR control is got ready, add viral crude extract, concentration is decremented to 1ng/mL from 10 times of gradients of 100mg/mL, each concentration gradient is got 25 μ L and is carried out immunosorption, from volume is to draw 2 μ L the reverse transcription product of 10 μ L to carry out real-time fluorescence PCR and detect, therefore corresponding to the crude extract of 100mg/mL concentration, the amount of the RNA that needs in the actual detected process only comes from 500ug leaf texture.
Cowpea severe mosaic virus (CPSMV) immunocapture-real-time fluorescence-detected leaf texture of PCR quality can be from 500ug to 500pg, corresponding C
TValue is followed successively by 19.46,20.86,23.93,27.33,30.33,33.624 and 34, positive control C
TValue is 16.32.Therefore, the sensitivity of IC-RT-Realtime PCR detection CPSMV can reach 500pg leaf texture.
SEQUENCE?LISTING
＜110〉Propagation and Food Test Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau
＜120〉a kind of molecule detection method for cowpea severe mosaic virus
<130>JSJYJYJ091
<160>3
<170>PatentIn?version?3.3
<210>1
<211>20
<212>DNA
<213>Artificial
<220>
<223>CPSMVF
<400>1
ggtcaatccc?ggcattattg 20
<210>2
<211>20
<212>DNA
<213>Artificial
<220>
<223>CPSMVR
<400>2
ggcttctgca?ggtgttccaa 20
<210>3
<211>27
<212>DNA
<213>Artificial
<220>
<223>CPSMVPro
<400>3
tgtagcacaa?tcagggcaaa?cacagca 27
Claims (3)
1. molecule detection method for cowpea severe mosaic virus, concrete steps are as follows:
(1) preparation of sample virus crude extract: in sample to be checked, add extraction buffer,, get supernatant liquor, be viral crude extract through grinding and centrifugal;
(2) preparation of immunocapture PCR pipe: get cowpea severe mosaic virus coated antibody after the dilution and add in the PCR pipe and hatch, after the lavation buffer solution washing, add the described viral crude extract of step 1,4 ℃ of refrigerator overnight incubation, the washing back is air-dry, promptly obtains immunocapture PCR pipe;
(3) cowpea severe mosaic virus RNA discharges and reverse transcription in the immunocapture PCR pipe: the cowpea severe mosaic virus RNA in the described immunocapture PCR of release steps (2) pipe and be template with it, with sequence be: the CPSMVR of 5 '-GGCTTCTGCAGGTGTTCCAA-3 ' is that primer carries out reverse transcription reaction, obtains reverse transcription product;
(4) real-time fluorescence PCR reaction: with step (3) gained reverse transcription product is template, forward primer CPSMVF sequence: 5 '-GGTCAATCCCGGCATTATTG-3 ', the sequence of reverse primer CPSMVR: 5 '-GGCTTCTGCAGGTGTTCCAA-3 ', the sequence of Taq Man probe CPSMVPro: 5 ' FAM-TGTAGCACAATCAGGGCAAACACAGCA-TAMRA 3 ', carry out the real-time fluorescence PCR reaction;
(5) interpretation of result: after the described real-time fluorescence PCR reaction of step 4 finishes, utilize real-time fluorescence PCR instrument analysis software, the collection of illustrative plates of amplification is analyzed, as initial cycle number (C
T)≤35 o'clock judge that the real-time fluorescence PCR reaction is positive, and the viral sample that expression is detected is a cowpea severe mosaic virus.
2. molecule detection method for cowpea severe mosaic virus according to claim 1, it is characterized in that cowpea severe mosaic virus RNA in the described immunocapture PCR of step (3) pipe discharges and the detailed process of reverse transcription is: in described immunocapture PCR pipe, add viral RNA and discharge liquid, handle 5min for 95 ℃ then, rapidly after the cooling, add following material again and carry out reverse transcription: 5 * Buffer of 2 μ L, 0.5 the RT Enzyme Mix I of μ L, response procedures is: 42 ℃ of reaction 60min; The concentration that consists of 0.5 μ L that described viral RNA discharges liquid is the ddH of the no RNA enzyme of the reverse primer CPSMVR of 10 μ mol/L and 7.0 μ L
2O.
3. molecule detection method for cowpea severe mosaic virus according to claim 1, it is characterized in that the described real-time fluorescence PCR reaction system of step 4 contains: 2 * Premix Ex Taq of 10 μ L, concentration is CPSMVF and each 0.8 μ L of CPSMVR of 10 μ mol/L, 0.2 μ L concentration is the CPSMVPro of 10 μ mol/L, 2 μ L reverse transcription products, the ddH of 6.2 μ L
2O; The reaction conditions of described real-time fluorescence PCR: 94 ℃ of reaction 3min; 94 ℃ of reaction 15sec, 60 ℃ of reactions 60sec, totally 45 circulations.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140555A (en) * | 2011-04-07 | 2011-08-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN106222305A (en) * | 2016-08-24 | 2016-12-14 | 福建省农业科学院果树研究所 | A kind of blue berry shock viruses molecule detection kit and detection method thereof |
| CN108486283A (en) * | 2018-03-26 | 2018-09-04 | 福建省农业科学院果树研究所 | A kind of cordate telosma mosaic virus Molecular Detection kit and detection method |
| CN112410448A (en) * | 2020-12-17 | 2021-02-26 | 中华人民共和国金陵海关 | Pseudomonas syringae pea pathogenic variety droplet type digital PCR molecular detection method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100421540C (en) * | 2005-04-14 | 2008-10-01 | 江汉大学 | Chemical method for controlling and cultivating high yield high grade asparagus bean |
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2009
- 2009-12-16 CN CN2009102630989A patent/CN101818211B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102140555A (en) * | 2011-04-07 | 2011-08-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN102140555B (en) * | 2011-04-07 | 2014-09-03 | 厦门出入境检验检疫局检验检疫技术中心 | Degenerate primers for universal reverse transcription-polymerase chain reaction (RT-PCR) detection of cowpea mosaic virus and fabavirus, detection method and application thereof |
| CN106222305A (en) * | 2016-08-24 | 2016-12-14 | 福建省农业科学院果树研究所 | A kind of blue berry shock viruses molecule detection kit and detection method thereof |
| CN108486283A (en) * | 2018-03-26 | 2018-09-04 | 福建省农业科学院果树研究所 | A kind of cordate telosma mosaic virus Molecular Detection kit and detection method |
| CN112410448A (en) * | 2020-12-17 | 2021-02-26 | 中华人民共和国金陵海关 | Pseudomonas syringae pea pathogenic variety droplet type digital PCR molecular detection method |
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| CN101818211B (en) | 2012-03-28 |
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