CN1975427B - Sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit, preparation thereof and detecting method - Google Patents
Sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit, preparation thereof and detecting method Download PDFInfo
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Abstract
The invention discloses double-antibody sandwich ELSA regent box and the method for the Cymbidiummosaicvirus. The box is made up of the box body, every using liquid and the enzyme labeled board. The preparation of the regent box is simple, quick, safe and reliable. The sample can be used little and it can be detected in a little time. Comparing to other serology detecting method, the method is simple, quick, sensitivity and high specialty, so it can be used widely in the base unit.
Description
Technical field
The present invention relates to detect kit and the preparation and the detection method of cymbidium mosaic virus, particularly relate to sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit and prepare the method for this kit and the detection method that this kit of application carries out cymbidium mosaic virus double antibodies sandwich enzyme joint inspection survey.
Background technology
(Odntoglossum ringspot virus ORSV) belongs to Tobamovirus (Tobamovirus) to cymbidium mosaic virus, is one of two kinds of main viruses of harm orchid, distributes all over the world main harm tooth orchid, sword-leaved cymbidium etc.In a single day orchid infects ORSV, and its blade often produces yellow striped or irregular chlorisis color spot piece, diseased plant flower portion even the deformity or the spot that fades occur, and the red colour system flower spot that fades is particularly evident, the ornamental value of orchid and economic worth is caused have a strong impact on.China's orchid aboundresources shows but study, and no matter is the potted plant living orchid in ground or the orchid of group training, and this virosis is all quite general. in view of the harmfulness of ORSV to China's orchid industry, it is very important to study its detection technique.
Plant virus detects enzyme linked immunosorbent assay (ELISA) (ELISA) method commonly used; EIA enzyme immunoassay is the technology that the specificity that antigen-antibody combines is combined with the efficient catalytic property of enzyme; Have characteristics such as highly sensitive, high specificity, but operation steps is many; Length consuming time, and need relatively costly enzyme labelled antibody and 96 hole ELISA Plates.
It is the detection method that on the enzyme-linked immunosorbent assay for measuring basis, grows up that the joint inspection of double antibodies sandwich enzyme is surveyed; Be suitable for detecting comparatively sensitive, the reliable and easy and simple to handle detection technique of a large amount of samples, but do not see as yet that at present the coupling of relevant double antibodies sandwich enzyme is in the bibliographical information that detects cymbidium mosaic virus and be used to detect this viral double antibodies sandwich elisa kit for detecting product.
Summary of the invention
The kit that the object of the present invention is to provide easy, quick, safe, reliable and economical and practical cymbidium mosaic virus double antibodies sandwich enzyme joint inspection to survey.
Another object of the present invention is to provide the method for preparing sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit.
A purpose more of the present invention is to provide the sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit that utilizes preparation to carry out the detection method that cymbidium mosaic virus is surveyed in the joint inspection of double antibodies sandwich enzyme.
For realizing above-mentioned purpose, technical solution of the present invention is:
Sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit mainly by box body, is located at various with liquid and ELISA Plate composition in the box body, variously mainly comprises height tire antiserum, labelled antibody, positive and negative sample, various damping fluids and developer with liquid.
Described positive is the cymbidium mosaic virus of purifying.
Described negative sample grinds the solution of healthy leaf preparation for sealing buffer solution.
Described damping fluid comprises:
Encapsulate damping fluid: 20-80mmol/L pH9.6 carbonate solution;
Sealing damping fluid: 20-100mmol/L pH7.4PBS+1-5% skimmed milk power;
Lavation buffer solution: 50-200mmol/L pH7.4PBS+0.05-0.3%Tween20;
Substrate buffer solution: 5-20%pH9.8 diethanolamine solution;
Stop buffer: 1-5mol/L sulfuric acid solution;
Described developer is a 0.05-0.5mg/ml pH9.8PNP-Na solution.
The preparation method of described sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit, it mainly comprises the following steps:
1, the extraction of cymbidium mosaic virus and purifying: after getting the homogenate of the sick leaf adding of 100g datura 100ml0.5M citrate buffer solution (pH6.5), add the 100ml chloroform and stirred 30 minutes.At Beckman10, centrifugal 20 minutes of 000rpm (JA-10).Get the NaCl that supernatant adds 4%PEG6000 and 0.3mol, left standstill after the stirring 1 hour.At Beckman10, centrifugal 20 minutes of 000rpm (JA-10) abandons supernatant, fully repeats the PEG deposition one time behind the dissolution precipitation with 0.5M citrate buffer solution (pH6.5).After getting for the second time post precipitation and suspending with the 5mM borate buffer, at Beckman10, centrifugal 20 minutes of 000rpm (JA-10) gets supernatant in ultracentrifuge use P42A rotary head 40, centrifugal 2 hours of 000rpm.Get deposition and suspend again, carry out differential centrifugation again one time, get deposition at last and dissolve with an amount of 0.01M PB (pH7.0) with 0.01M PB (pH7.0).If impurity is too many, carry out differential centrifugation again one time.Get thick purified virus and place 10%
-40% sucrose pad gradient is used P42A rotary head 40, centrifugal 2 hours of 000rpm in ultracentrifuge.Collect viral band, use P42A rotary head 40 in ultracentrifuge once more, centrifugal 2 hours deposition virus of 000rpm is dissolved among the 0.01M PB (pH7.0) at last, places-40 ℃ of preservations subsequent use.
2, sero-fast preparation and purifying: select for use male rabbit to do immune animal, the purified virus that adds the emulsification of equivalent FreundShi Freund is done immunogene, adopts three intramuscular injection and twice intravenous immune rabbit.The viral amount of per injection is respectively 0.5mg, 0.75mg, 1mg, 1.5mg and 2mg, and be 7 days interval time, and last injection back blood sampling in 7-10 days 2 times is separated out serum and tired with agar double immunodiffusion method mensuration; Combine DEAE ion-exchange chromatography antibody purification through the sad precipitation method.Regulate antiserums to pH4.8 with 2 times of volume 0.1M ammonium acetates, press 0.75ml sad/1ml serum adds caprylic acid, mixes and stirs 1 hour, centrifugal 30 minutes of 5000rpm gets supernatant, places 10mM Tris-Cl pH8.5 damping fluid to spend the night bag filter, the desalination of dialysing.Get and dialyse completely that solution is splined in the ion exchange column, with 10mMTris-HCl pH8.5 damping fluid 5ml/min flushing 15 minutes, again with containing 0
-The 10mM Tris-HCl pH8.5 buffer concentration gradient of 1M NaCl is carried out wash-out, collects the eluting peak part, concentrates dialysis, obtains the cymbidium mosaic virus polyclonal antibody of purifying.
3 antibody labelings: alkali phosphatase enzyme mark is arrived antibody purification through the glutaraldehyde two step method.Every 1mg antibody adds 0.1% glutaraldehyde 0.05ml, leaves standstill 1 hour, places bag filter to remove unnecessary glutaraldehyde.Add the 0.5mg alkaline phosphatase, mixing be placed on 4 ℃ subsequent use.
4, the preparation of the positive, negative sample:
Positive is the cymbidium mosaic virus virus of purifying, and concentration is in 0.1 μ g-100 μ g/ml scope; Negative sample grinds the solution of healthy leaf preparation for sealing buffer solution.
5, various preparations with liquid:
Encapsulate damping fluid: 20-80mmol/L pH9.6 carbonate solution;
Sealing damping fluid: 20-100mmol/L pH7.4PBS+1-5% skimmed milk power;
Lavation buffer solution: 50-200mmol/L pH7.4PBS+0.05-0.3%Tween20;
Substrate buffer solution: 5-20%pH9.8 diethanolamine solution;
Stop buffer: 1-5mol/L sulfuric acid solution;
Developer is a 0.05-0.5mg/ml pH9.8PNP-Na solution.
The detection method of described sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit joins detection method for the double antibodies sandwich enzyme, and it may further comprise the steps:
1) encapsulate: encapsulate damping fluid with carbonate, with ORSV polyclone labelled antibody antibody dilution to 1～10ug/ul, in each reacting hole of ELISA Plate, every hole 100ul, was hatched 2 hours by 37 ℃;
2) sealing: the coating buffer that inclines, the confining liquid of skimmed milk power, every hole 100ul was hatched 1 hour for 37 ℃, then with lavation buffer solution washing three times;
3) application of sample: add sample to be detected, every hole 100ul, and set up feminine gender, the positive and blank, hatched 1 hour for 37 ℃, then with lavation buffer solution washing three times;
4) mark spike: add enzyme labelled antibody, every hole 100ul was hatched 1 hour for 37 ℃, then with lavation buffer solution washing three times;
5) colour developing: add the colour developing liquid with the preparation of 5-20%pH9.8 diethanolamine substrate buffer solution, every hole 100ul was hatched 30 minutes for 37 ℃;
6) cessation reaction is judged with detection: add the stop buffer of 1-5mol/L sulfuric acid, every hole 50ul measures the OD405 value on ELIASA.
After adopting such scheme, the present invention is by box body, is located at various in the box body and is assembled into economical and practical detection kit with liquid, ELISA Plate.The preparation method of kit is easy, quick, safe, reliable and economical and practical.Amount of samples is few; Just can accomplish detection in the short period of time; And the result is easy to preserve; Use this detection kit carry out the double antibodies sandwich enzyme join method that joint inspection surveys compare with other serologic test methods have simple, fast, characteristics such as sensitivity, high specificity, and have higher repeatability, more advantage such as economical and practical, so that apply in grass-roots unit.
Embodiment
The main agents that the present invention is used: the cymbidium mosaic virus polyclonal antibody is Xiamen Overseas Chinese Subtropical Plants Introduction Garden's preparation; PEG6000,2 mercapto ethanol, Triton-100, bovine serum albumin(BSA) (BSA) are available from Sigma company; Alkaline phosphatase, PNPP-Na, DEAE ion-exchange filling material, IgG affinity chromatography filling material are available from Pierce company; Employed other conventional medicines and reagent are homemade AR in the test.
One, kit
Sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit mainly by box body, is located at various with liquid and ELISA Plate composition in the box body, variously mainly comprises height tire antiserum, labelled antibody, positive and negative sample, various damping fluids and developer with liquid.
Positive is the cymbidium mosaic virus of purifying.
Negative sample grinds the solution of healthy leaf preparation for sealing buffer solution.
Damping fluid comprises:
Encapsulate damping fluid: 50mmol/L pH9.6 carbonate solution;
Sealing damping fluid: 50mmol/L pH7.4PBS+1-5% skimmed milk power;
Lavation buffer solution: 100mmol/L pH7.4PBS+0.05-0.3%Tween20;
Substrate buffer solution: 10%pH9.8 diethanolamine solution;
Stop buffer: 2mol/L sulfuric acid solution;
Developer is a 0.1mg/ml pH9.8PNP-Na solution.
Two, preparation method
The preparation method of cymbidium mosaic virus detection kit of the present invention, it comprises the following steps:
1, the extraction of cymbidium mosaic virus and purifying: after getting the homogenate of the sick leaf adding of 100g datura 100ml0.5M citrate buffer solution (pH6.5), add the 100ml chloroform and stirred 30 minutes.At Beckman10, centrifugal 20 minutes of 000rpm (JA-10).Get the NaCl that supernatant adds 4%PEG6000 and 0.3mol, left standstill after the stirring 1 hour.At Beckman10, centrifugal 20 minutes of 000rpm (JA-10) abandons supernatant, fully repeats the PEG deposition one time behind the dissolution precipitation with 0.5M citrate buffer solution (pH6.5).After getting for the second time post precipitation and suspending with the 5mM borate buffer, at Beckman10, centrifugal 20 minutes of 000rpm (JA-10) gets supernatant in ultracentrifuge use P42A rotary head 40, centrifugal 2 hours of 000rpm.Get deposition and suspend again, carry out differential centrifugation again one time, get deposition at last and dissolve with an amount of 0.01M PB (pH7.0) with 0.01M PB (pH7.0).If impurity is too many, carry out differential centrifugation again one time.Get thick purified virus and place 10%
-40% sucrose pad gradient is used P42A rotary head 40, centrifugal 2 hours of 000rpm in ultracentrifuge.Collect viral band, use P42A rotary head 40 in ultracentrifuge once more, centrifugal 2 hours deposition virus of 000rpm is dissolved among the 0.01M PB (pH7.0) at last, places-40 ℃ of preservations subsequent use.
2, sero-fast preparation and purifying: select for use male rabbit to do immune animal, the purified virus that adds the emulsification of equivalent FreundShi Freund is done immunogene, adopts three intramuscular injection and twice intravenous immune rabbit.The viral amount of per injection is respectively 0.5mg, 0.75mg, 1mg, 1.5mg and 2mg, and be 7 days interval time, and last injection back blood sampling in 7-10 days 2 times is separated out serum and tired with agar double immunodiffusion method mensuration; Combine DEAE ion-exchange chromatography antibody purification through the sad precipitation method.Regulate antiserums to pH4.8 with 2 times of volume 0.1M ammonium acetates, press 0.75ml sad/1ml serum adds caprylic acid, mixes and stirs 1 hour, centrifugal 30 minutes of 5000rpm gets supernatant, places 10mM Tris-Cl pH8.5 damping fluid to spend the night bag filter, the desalination of dialysing.Get and dialyse completely that solution is splined in the ion exchange column, with 10mMTris-HCl pH8.5 damping fluid 5ml/min flushing 15 minutes, again with containing 0
-The 10mM Tris-HClpH8.5 buffer concentration gradient of 1M NaCl is carried out wash-out, collects the eluting peak part, concentrates dialysis, obtains the cymbidium mosaic virus polyclonal antibody of purifying.
3, antibody labeling: alkali phosphatase enzyme mark is arrived antibody purification through the glutaraldehyde two step method.Every 1mg antibody adds 0.1% glutaraldehyde 0.05ml, leaves standstill 1 hour, places bag filter to remove unnecessary glutaraldehyde.Add the 0.5mg alkaline phosphatase, mixing be placed on 4 ℃ subsequent use.
4, the preparation of the positive, negative sample
Positive is the cymbidium mosaic virus virus of purifying, and concentration is in 0.1 μ g-100 μ g/ml scope; Negative sample grinds the solution of healthy leaf preparation for sealing buffer solution.
5, various preparations with liquid
Encapsulate damping fluid: 50mmol/L pH9.6 carbonate solution;
Sealing damping fluid: 50mmol/L pH7.4PBS+1-5% skimmed milk power;
Lavation buffer solution: 100mmol/L pH7.4PBS+0.05-0.3%Tween20;
Substrate buffer solution: 10%pH9.8 diethanolamine solution;
Stop buffer: 2mol/L sulfuric acid solution;
Developer is a 0.1mg/ml pH9.8PNP-Na solution.
Three, detection method embodiment
The detection method of sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit joins detection method for the double antibodies sandwich enzyme, and it may further comprise the steps:
1) encapsulate: encapsulate damping fluid with 50mmol/L pH9.6 carbonate, to 10ug/ul, in each reacting hole of ELISA Plate, every hole 100ul, is hatched 2h by 37 ℃ with 0RSV polyclone labelled antibody antibody dilution;
2) sealing: the coating buffer that inclines, the confining liquid of adding 50mmol/L pH7.4PBS+1% skimmed milk power, every hole 100ul is hatched 1h for 37 ℃, and then the lavation buffer solution with 100mmol/L pH7.4PBS+0.1%Tween20 washs three times;
3) application of sample: add sample to be detected, every hole 100ul, and set up feminine gender, the positive and blank, and hatch 1h for 37 ℃, then the lavation buffer solution with 100mmol/L pH7.4PBS+0.1%Tween20 washs three times;
4) mark spike: add enzyme labelled antibody, every hole 100ul is hatched 1h for 37 ℃, and then the lavation buffer solution with 100mmol/L pH7.4PBS+0.1%Tween20 washs three times;
5) colour developing: add the colour developing liquid with the preparation of 10%pH9.8 diethanolamine substrate buffer solution, every hole 100ul is hatched 30min for 37 ℃;
6) cessation reaction is judged with detection: add the stop buffer of 2mol/L sulfuric acid, every hole 50ul measures the OD405 value on ELIASA.
Criterion is surveyed in the joint inspection of double antibodies sandwich enzyme:
Testing sample is by the negative OD405 of OD405/>2 be judged to be the positive, < 2 are judged to be feminine gender to the negative OD405 of OD405/.
Testing sample qualitatively judges the yin, yang of testing sample testing result by above standard;
For check double antibodies sandwich elisa kit for detecting effect, 40 parts of orchid samples have been detected with this method.
Testing result is following:
Annotate: "+" represents positive; "-" represents negative
31 parts of samples by the RT-PCR detection positive, DIBA detects 30 parts of positive, 1 part of feminine gender;
9 parts of samples by RT-PCR detection feminine gender, DIBA detects negative sample part, 1 part of positive;
Can be known by testing result: the susceptibility of DIBA is 96.77%; Specificity 88.89%; Detection accuracy is 95%.
Claims (2)
1. sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit; Mainly by box body, be located at various in the box body with liquid and ELISA Plate composition, variously mainly comprise height tire antiserum, labelled antibody, positive and negative sample with liquid; Various damping fluids and developer is characterized in that:
The described height antiserum of tiring: select for use male rabbit to do immune animal; The purified virus that adds the emulsification of equivalent FreundShi Freund is done immunogene; Adopt three intramuscular injection and twice intravenous immune rabbit, the amount of per injection virus is respectively 0.5mg, 0.75mg, 1mg, 1.5mg and 2mg, and be 7 days interval time; Last injection back blood sampling in 7-10 days 2 times is separated out serum and is tired with agar double immunodiffusion method mensuration; Combine DEAE ion-exchange chromatography antibody purification through the sad precipitation method, regulate antiserums to pH4.8 with 2 times of volume 0.1M ammonium acetates, press 0.75ml sad/1ml serum adding caprylic acid; Mix and stirred 1 hour, centrifugal 30 minutes of 5000rpm gets supernatant; Place 10mM Tris-Cl pH8.5 damping fluid to spend the night bag filter, the dialysis desalination is got and is dialysed completely that solution is splined in the ion exchange column; With 10mM Tris-HClpH8.5 damping fluid 5ml/min flushing 15 minutes, carry out wash-out with the 10mMTris-HCl pH8.5 buffer concentration gradient that contains 0-1M NaCl again, collect the eluting peak part; Concentrate dialysis, obtain the cymbidium mosaic virus polyclonal antibody of purifying;
Described labelled antibody: alkali phosphatase enzyme mark is arrived antibody purification through the glutaraldehyde two step method; Every 1mg antibody adds 0.1% glutaraldehyde 0.05ml, leaves standstill 1 hour, places bag filter to remove unnecessary glutaraldehyde; Add the 0.5mg alkaline phosphatase, mixing be placed on 4 ℃ subsequent use;
Described positive is the cymbidium mosaic virus of purifying; Concentration is in 0.1 μ g-100 μ g/ml scope;
Described negative sample grinds the solution of healthy leaf preparation for sealing buffer solution;
Described damping fluid comprises:
Encapsulate damping fluid: 20-80mmol/L pH9.6 carbonate solution;
Sealing damping fluid: 20-100mmol/L pH7.4 PBS+1-5% skimmed milk power;
Lavation buffer solution: 50-200mmol/L pH7.4 PBS+0.05-0.3% Tween20;
Substrate buffer solution: 5-20% pH9.8 diethanolamine solution;
Stop buffer: 1-5mol/L sulfuric acid solution;
Described developer is a 0.05-0.5mg/ml pH9.8 PNP-Na solution.
2. the preparation method of sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit as claimed in claim 1, it is characterized in that: described preparation method mainly comprises following preparation process:
1), the extraction and the purifying of cymbidium mosaic virus: after getting the sick leaf of 100g datura and adding 100ml0.5M citrate buffer solution pH6.5 homogenate, add the 100ml chloroform and stirred 30 minutes, on Beckman JA-10 hydro-extractor 10, centrifugal 20 minutes of 000rpm; Get the NaCl that supernatant adds 4%PEG6000 and 0.3mol, left standstill after the stirring 1 hour, on Beckman JA-10 hydro-extractor 10; Centrifugal 20 minutes of 000rpm abandons supernatant, with repeating the PEG deposition behind pH6.5, the abundant dissolution precipitation of 0.5M citrate buffer solution one time; Get for the second time post precipitation with the suspension of 5mM borate buffer after, on Beckman JA-10 hydro-extractor 10, centrifugal 20 minutes of 000rpm; Get supernatant and use P42A rotary head 40 in ultracentrifuge, centrifugal 2 hours of 000rpm gets deposition and suspends again with pH7.0,0.01M PB; Carry out differential centrifugation again one time, get deposition at last with an amount of pH7.0,0.01M PB dissolving, if impurity is too many; Carry out differential centrifugation again one time, get thick purified virus and place 10%-40% sucrose pad gradient, use P42A rotary head 40 in ultracentrifuge; Centrifugal 2 hours of 000rpm collects viral band, uses P42A rotary head 40 in ultracentrifuge once more; Centrifugal 2 hours deposition virus of 000rpm is dissolved among pH7.0, the 0.01M PB at last, places-40 ℃ of preservations subsequent use;
2), sero-fast preparation and purifying: select for use male rabbit to do immune animal; The purified virus that adds the emulsification of equivalent FreundShi Freund is done immunogene; Adopt three intramuscular injection and twice intravenous immune rabbit, the amount of per injection virus is respectively 0.5mg, 0.75mg, 1mg, 1.5mg and 2mg, and be 7 days interval time; Last injection back blood sampling in 7-10 days 2 times is separated out serum and is tired with agar double immunodiffusion method mensuration; Combine DEAE ion-exchange chromatography antibody purification through the sad precipitation method, regulate antiserum to pH4.8 with 2 times of volumes, 0.1 M ammonium acetate, press 0.75ml sad/1ml serum adding caprylic acid; Mix and stirred 1 hour, centrifugal 30 minutes of 5000rpm gets supernatant; Place 10mM Tris-Cl pH8.5 damping fluid to spend the night bag filter, the dialysis desalination is got and is dialysed completely that solution is splined in the ion exchange column; With 10mMTris-HCl pH8.5 damping fluid 5ml/min flushing 15 minutes, carry out wash-out with the 10mM Tris-HCl pH8.5 buffer concentration gradient that contains 0-1M NaCl again, collect the eluting peak part; Concentrate dialysis, obtain the cymbidium mosaic virus polyclonal antibody of purifying;
3), antibody labeling: alkali phosphatase enzyme mark is arrived antibody purification through the glutaraldehyde two step method; Every 1mg antibody adds 0.1% glutaraldehyde 0.05ml, leaves standstill 1 hour, places bag filter to remove unnecessary glutaraldehyde; Add the 0.5mg alkaline phosphatase, mixing be placed on 4 ℃ subsequent use;
4), the preparation of the positive, negative sample:
Positive is the cymbidium mosaic virus virus of purifying, and concentration is in 0.1 μ g-100 μ g/ml scope; Negative sample grinds the solution of healthy leaf preparation for sealing buffer solution;
5), various preparations with liquid:
Encapsulate damping fluid: 20-80mmol/L pH9.6 carbonate solution;
Sealing damping fluid: 20-100mmol/L pH7.4 PBS+1-5% skimmed milk power;
Lavation buffer solution: 50-200mmol/L pH7.4 PBS+0.05-0.3% Tween20;
Substrate buffer solution: 5-20% pH9.8 diethanolamine solution;
Stop buffer: 1-5mol/L sulfuric acid solution;
Developer is a 0.05-0.5mg/ml pH9.8 PNP-Na solution.
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| CN2006101644336A CN1975427B (en) | 2006-02-10 | 2006-12-08 | Sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit, preparation thereof and detecting method |
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| CN2006101644336A CN1975427B (en) | 2006-02-10 | 2006-12-08 | Sword-leaved cymbidium leaf virus double anti sandwich enzyme-linked assay kit, preparation thereof and detecting method |
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| CN101893633A (en) * | 2010-08-06 | 2010-11-24 | 扬州大学 | Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method for detecting porcine parvovirus |
| CN102621306B (en) * | 2012-03-22 | 2014-09-10 | 中国人民解放军总医院 | Pepsin enzyme-linked immunoassay detection kit |
| CN102676695B (en) * | 2012-04-16 | 2014-03-26 | 杭州师范大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit capable of detecting CymMV (Cymbidium Mosaic Virus) and ORSV (Odontoglossum Ringspot Virus) simultaneously and method thereof |
| CN104007211A (en) * | 2014-06-13 | 2014-08-27 | 江南大学 | Method for extracting plant viruses from plant tissue and authenticating plant viruses through MALDI-TOF-MS |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1611940A (en) * | 2004-04-15 | 2005-05-04 | 云南省农业科学院 | PVX Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN1611941A (en) * | 2004-04-15 | 2005-05-04 | 云南省农业科学院 | PVY Yunnan isolate TAS-ELISA test kit and its preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1611940A (en) * | 2004-04-15 | 2005-05-04 | 云南省农业科学院 | PVX Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN1611941A (en) * | 2004-04-15 | 2005-05-04 | 云南省农业科学院 | PVY Yunnan isolate TAS-ELISA test kit and its preparing method |
Non-Patent Citations (2)
| Title |
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| 明艳林 李梅 郑国华.《建兰花叶病毒研究进展(综述)》.《福建农业学报》.2005,第20卷(第1期),30-33. * |
| 明艳林 郑国华 李梅.《建兰花叶病毒厦门分离物的鉴定及其抗血清的制备与应用》.《植物病理学报 》.2005,第35卷(第4期),366-369,具体参见第367页第1.7、1.8节,第368页2.5,2.6节. * |
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