CN102928598A - Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof - Google Patents
Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof Download PDFInfo
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Abstract
The invention disclose a dot- ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting the presence of the tomato yellow leaf curl virus in a plant as well as a reagent kit and an application thereof. A monoclonal antibody prepared for tomato yellow leaf curl virus coat protein is utilized to build the dotenzyme-Linked immunosorbent assay (dot-ELISA) method and the tissue printing ELISA method with optimal proportions, and the rapid-detection reagent kit is developed. The dot- ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting the presence of the tomato yellow leaf curl virus in a plant as well as the reagent kit and the application thereof are suitable for such solanaceae plants as tomato, capsicum, eggplant, tobacco, night shade and jimson weed; the situation of the presence of tomato yellow leaf curl virus in field vegetable samples is examined to detect the incidence of the virus disease and evaluate the occurrence and distribution and the prevalence trend of the tomato yellow leaf curl virus under the field condition; and the detection method has high sensitivity and good specificity, needs short time, is low in cost, and provides a technical support for rapid and large-scale detection of tomato yellow leaf curl virus.
Description
Technical field
Field under the present invention is the plant protection field, relates in particular to the dot-ELISA and the Application and Development of organizing trace ELISA method for quick and detection kit thereof of tomato tomato yellow leaf curl virus.
Background technology
Tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) causes on the tomato leaf volume or last volume, blade yellow, vein evagination, plant to be downgraded, and causes the tomato total crop failure when serious.It is reported, cause the cause of disease of tomato in China tomato yellow leaf curl China to have 15 kinds, what wherein occur that area is the widest, harm is the heaviest in China is tomato yellow leaf curl virus.Tomato yellow leaf curl virus belong to geminivirus infection section (
Geminiviridae) Begomovirus (
Begomovirus) virus, this belong to virus under field conditions (factors) can only by Bemisia tabaci (
Bemisia tabaci) propagate in the persistence mode.
From Shanghai in 2006 and Zhejiang successively since tomato is found tomato yellow leaf curl virus, the tomato leaf curl that this virus causes comes at the East China rapid spread, popular outburst on the tomato in a plurality of provinces such as Beijing, Shandong, Jiangsu, Anhui, Zhejiang, Hainan, Fujian, Yunnan, Guangdong, Guangxi, Xinjiang.Because the business activities such as the variation of global warming and agricultural development general layout, particularly nursery stock allocation and transportation is day by day frequent, so that the tomato yellow leaf curl evil spreads rapidly and spreads from south orientation north in China and comes, tomato production is consisted of grave danger.According to incompletely statistics, the year of tomato in China tomato yellow leaf curl China virus disease area occurs and surpass 1,000,000 mu, year economic loss at least 20 hundred million Renminbi.Present this disease has become tomato in China and has produced important restraining factors.
Because the tomato yellow leaf curl virus disease is in upward period, breaks out the situation sternness China, early monitoring and the early warning technology of therefore strengthening tomato yellow leaf curl virus are very urgent.But because tomato yellow leaf curl virus only is present in plant phloem, and the content of virus is lower in the diseased plant, causes conventional virosis detection method to be difficult to use.At present, the detection technique of tomato yellow leaf curl virus mainly relies on the means such as pcr amplification, nucleic acid hybridization and serology.Although PCR detection technique sensitivity is high, but need through extracting genome DNA---pcr amplification---electrophoretic analysis even these steps of genome sequence determination, loaded down with trivial details, the consuming time length of experimental implementation process and this technology place one's entire reliance upon PCR instrument and business-like reagent and to cause experimentation cost higher are unfavorable for the extensive detection of field virosis.Round pcr also is difficult for promoting to the layman because of reasons such as false positive height in addition.Though nucleic acid hybridization technique high specificity, experimental implementation are more professional and loaded down with trivial details, except highly relying on the instrument reagent is also had high requirements.Although the radioactive isotope sensitivity that tradition is used is high, this reagent all has certain harm to human body and environment, is unfavorable for environmental protection.Though the no radioactivity pollute such as the substituting product of releasing recently such as digoxin reagent but expensive is from the angle of economy and practicality so that nucleic acid hybridization technique is difficult to be applied to the detection of a large amount of viral sample in field.The serology detection technique of setting up take monoclonal antibody as core is comparatively easy detection method, it has the advantages such as easy to operate, that sensitivity is high, but traditional serological method such as tri-antibody sandwich ELISA (TAS-ELISA), antigen coated ELISA(ACP-ELISA) etc. method need to be in 48 holes or 96 hole enzyme-linked reaction plates carry out, experimental procedure is many, each step reaction time is long, data are by the microplate reader device value of reading, and high to the instrument dependence, experiment material high in cost of production characteristics are unfavorable for the detection of extensive sample.In the patent of the former first to file of this seminar " secrete anti-tomato yellow leaf curl virus monoclonal antibody hybridoma cell strain and monoclonal antibody thereof use " (Chinese patent application number be 201210004197.7), embodiment is briefly mentioned dot-ELISA and Tissue-blot ELISA detection method, but utilize the method when reality is used, to find, the method is only suitable for detecting virus in a Plants (being tomato leaf), is not suitable for other plant; In addition, because the problem of plant sampling point and reagent proportioning causes viral recall rate to have much room for improvement.The Tissue-blot ELISA kit of particularly mentioning among the embodiment, tomato leaf consumption is many when using in the field, reagent dosage large, cost is higher.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, further optimizing on the basis of each experimental data, the dot-ELISA that detects the plant infection tomato yellow leaf curl virus is provided and organizes trace ELISA method and kit and application.
Detect the dot-ELISA rapid identification method of plant infection tomato yellow leaf curl virus, comprise the steps:
1) cellulose nitrate (NC) film is prepared: rule at NC film skin paper with pencil, make the NC film stamp the graticule line vestige, every lattice specification 1 * 1 cm makes marks the smooth double dish central authorities that put at NC film one jiao;
2) plant sample is processed: volume ratio 1:30 to 1:50(grams per milliliter by weight) ratio takes by weighing plant stem, and add 0.01 mol/L PBS plant tissue is ground to form the homogenate shape;
3) point sample: draw 2 ul plant supernatants with liquid-transfering gun and put the grid central authorities that pull;
4) sealing: behind the point sample, the NC film left standstill dry 10 minutes, use the ratio preparation that adds 100 mL PBST damping fluids (the 0.01 mol/L PBS that contains 0.05% Tween-20) in 5 g skimmed milk powers to obtain 5% skimmed milk power sealing, 37 ℃ left standstill 0.5-1 hour;
5) add primary antibodie: add Chinese patent application and number be the monoclonal antibody of 201210004197.7 tomato yellow leaf curl virus, the skimmed milk power 1:2000 of this monoclonal anti body and function 5% doubly dilutes, and hatches 1 hour for 37 ℃;
6) washing: abandon liquid, the NC film shakes wash-out 3 times with the PBST damping fluid, each 3 minutes;
7) add ELIAS secondary antibody: abandon eluent, the sheep anti-mouse igg two that adds alkali phosphatase enzyme mark is anti-, and the skimmed milk power 1:8000 of two anti-usefulness 5% doubly dilutes, and hatches 1 hour for 37 ℃;
8) washing: abandon liquid, the NC film shakes wash-out 5 ~ 6 times with the PBST damping fluid, each 5 minutes;
9) colour developing: chromogenic substrate NBT 66 ul and BCIP 33 ul(S3771 Promega) be added to mixing in the 10 ml substrate buffer solutions, put into substrate nitrite ion reaction 10 minutes after washed NC film blots with filter paper;
10) cessation reaction: treat that sample on the NC film presents purple and abandon the nitrite ion cessation reaction when negative control does not develop the color;
11) record result: ddH
2O soaks the NC film, dries rear record result.
That detects the plant infection tomato yellow leaf curl virus organizes trace ELISA method for quick, comprises the steps:
1) the NC film is prepared: rule at NC film skin paper with pencil, make the NC film stamp the graticule line vestige, every lattice specification 1 * 1 cm makes marks the smooth double dish central authorities that put at NC film one jiao;
2) plant sample is processed and point sample: with blade cuts plant petiole, make otch concordant, by placing the grid central authorities that pull 3 ~ 5 seconds, each plant sample repeats aforesaid operations step once with cut-out section; In the operating process, plant sample of every cutting is changed blade or with the alcohol swab blade of sterilizing;
3) sealing: behind the point sample, the NC film left standstill dry 10 minutes, use the ratio preparation that adds 100 mL PBST damping fluids (the 0.01 mol/L PBS that contains 0.05% Tween-20) in 5 g skimmed milk powers to obtain 5% skimmed milk power sealing, 37 ℃ left standstill 0.5-1 hour;
4) add primary antibodie: add Chinese patent application and number be the monoclonal antibody of 201210004197.7 tomato yellow leaf curl virus, the skimmed milk power 1:2000 of monoclonal anti body and function 5% doubly dilutes, and hatches 1 hour for 37 ℃;
5) washing: abandon liquid, the NC film shakes wash-out 3 times with PBST, each 3 minutes;
7) add ELIAS secondary antibody: abandon eluent, the sheep anti-mouse igg two that adds alkali phosphatase enzyme mark is anti-, and the skimmed milk power 1:8000 of two anti-usefulness 5% doubly dilutes, and hatches 1 hour for 37 ℃;
8) washing: abandon liquid, the NC film shakes wash-out 5 ~ 6 times with the PBST damping fluid, each 5 minutes;
9) colour developing: chromogenic substrate NBT 66 ul and BCIP 33 ul(S3771 Promega) be added to mixing in the 10 ml substrate buffer solutions, put into substrate nitrite ion reaction 10 minutes after washed film blots with filter paper;
10) cessation reaction: treat that sample on the NC film presents purple and abandon the nitrite ion cessation reaction when negative control does not develop the color;
11) record result: ddH
2O soaks the NC film, dries rear record result.
Tomato yellow leaf curl virus dot-ELISA and organize trace ELISA detection kit comprises the steps:
1) kit main agents and material:
20 * concentrated PBS(0.2 mol/L PBS) 10 ml
Monoclonal antibody 0.1 ml
AP mark sheep anti-mouse igg two anti-0.02 ml
10 * concentrated confining liquid 10 ml
10 * concentrated antibody dilution, 20 ml
20 * concentrated cleaning solution, 70 ml
Sealer 15 g
Substrate buffer solution 70 ml
7 of NC films
NBT storing solution 0.4 ml
BCIP storing solution 0.2 ml
2) kit operation steps:
Organize trace ELISA step:
2.1) get tomato stem or petiole, use the blade crosscut, the square section is impressed 3-5 second at film immediately.Turn step 2.4.
The dot-ELISA step:
2.1) after the tomato stem weighs, volume ratio 1:30-1:50(grams per milliliter by weight) add 0.01 mol/L PBS and grind;
2.2) centrifugal 3 minutes of 5000 rpm;
2.3) get and check on the NC film on 2 ul;
2.4) drying at room temperature 10 minutes;
2.5) the NC film is immersed in the 1 * confining liquid that contains 5% sealer room temperature sealing 30-60 minute;
2.6) the NC film put into the monoclonal antibody incubated at room of doubly diluting with 1 * antibody diluent 1:2000 30-60 minute;
2.7) 1 * cleansing solution washes film 3-4 time, each 3 minutes;
2.8) the NC film put into the alkali phosphatase enzyme mark sheep anti-mouse igg two that doubly dilutes with 1 * antibody diluent 1:8000 anti-incubated at room 30-60 minute;
2.9) 1 * cleansing solution washes film 5-6 time, each 3 minutes;
2.10) chromogenic substrate NBT 66 ul and BCIP 33 ul(S3771 Promega) be added to mixing in the 10 ml substrate buffer solutions, putting into the substrate nitrite ion after washed film blots with filter paper reacts, treat the positive control colour developing obviously, and negative cessation reaction (developing the color 5-20 minute) during without any colour developing, namely in tap water rinsing once, visual inspection is also recorded the result.
3) kit application notice:
3.1) the NC film is positioned in the middle of 2 protection sheets, directly do not touch the NC film with hand;
3.2) one side that drips test sample on the NC film be the front, should be up in the whole experimentation;
3.3) dilution in the antibody 10 minutes before use;
3.4) the substrate nitrite ion is now with the current;
3.5) the positive contrast in NC film CK+ place; The negative contrast in NC film CK-place;
3.6) the kit temperature of reaction is 4-38 ℃, optimal reaction temperature is 37 ℃;
3.7) kit can detect 200 field samples.
4) holding conditions and storage life:
Kit keeps in Dark Place in 2-8 ℃, but antibody-20 ℃ freezing preservation is better, and NBT storing solution and BCIP storing solution must-20 ℃ of freezing preservations.Storage life: this keeping life is 6 months.
Described method is applicable to the host of tomato yellow leaf curl virus---plant of Solanaceae, and described plant of Solanaceae comprises tomato, capsicum, eggplant, tobacco, black nightshade, datura, and sampling point is stem or the petiole of plant.
Described method can be investigated the band poison situation of a large amount of samples in field, calculates tomato yellow leaf curl virus at the infection rate in field, and the field occurrence and distribution of assessment tomato yellow leaf curl and popular outburst trend are in order in time take prophylactico-therapeutic measures.
Beneficial effect of the present invention:
1) the invention provides the serological method of two kinds of fast detecting plant infection tomato yellow leaf curl virus---dot-ELISA and organize trace ELISA authentication method.
2) detection method applicability provided by the invention is wide.Tomato yellow leaf curl virus infects plant of Solanaceae mostly, and detection method provided by the invention energy effective application has enlarged the sensing range of plant in multiple plants of Solanaceae such as tomato, capsicum, eggplant, tobacco, black nightshade, daturas.
3) only be present in plant phloem for tomato yellow leaf curl virus, and the low characteristics of viral level in the plant, through strict contrast verification repeatedly, it is the high plant stem of tomato yellow leaf curl virus accumulation or petiole that the present invention proposes sampling point.The change of plant sampling point has improved the sensitivity that detects.
4) concentration of the various reagent in the detection kit provided by the invention and parameter proportioning are to optimize gained through up to a hundred the meticulous debugging of independent experiment.Such as the sampling amount that has reduced plant tissue, reduce the damage of the plant maximum economic benefit with the protection peasant; Improved the working concentration of monoclonal antibody, optimized the parameter proportioning of reagent etc., realized that on the basis of saving cost viral recall rate is the highest.The indices integrated use can reach the detection sensitivity height, high specificity, and weak point consuming time, cost is low, and have and wholely simple to operately fast professional skill is required low, experimentation does not need instrument, experiment material is easy to carry about with one, economic environmental protection, the advantage such as practical.A people only needs can finish in 3-4 hour the detection of hundreds of part plant sample, is beneficial to the large-scale promotion and application in field.
Description of drawings
Fig. 1-3 dot-ELISA grind away synoptic diagram;
Fig. 4 dot-ELISA method detects the result of field tomato sample;
Fig. 5-6 organizes trace ELISA operation chart;
Fig. 7 organizes trace ELISA method to detect the result of field tomato sample;
Fig. 8 field tomato sample is through the PCR the result;
Fig. 9 tomato yellow leaf curl virus (TYLCV) dot-ELISA and organize the external packing of trace ELISA detection kit;
Figure 10 tomato yellow leaf curl virus (TYLCV) dot-ELISA and material and the reagent of organizing trace ELISA detection kit;
Figure 11 utilizes the dot-ELISA method in the kit to detect the result of Hangzhou tomato sample;
Figure 12 utilizes the trace ELISA method of organizing in the kit to detect the result of Hangzhou tomato sample.
Embodiment
Embodiment one: utilize the method for dot-ELISA to detect the situation that infects of tomato yellow leaf curl virus in the tomato plant of field.
Gather tomato 14 strains with disease symptom from the Zhengzhou, henan farmland, detect according to the method for operating of dot-ELISA:
1) the NC film is prepared: rule at the outer paper of NC film with pencil, make the NC film stamp the graticule line vestige, every hole gauge lattice 1 * 1 cm makes marks in the lower right corner of NC film, the smooth double dish central authorities that put into;
2) ratio sample preparation: press 1:50(weight: volume, grams per milliliter) takes by weighing the tomato petiole, adds 0.01 mol/L PBS the tomato tissue is ground to form homogenate shape (Fig. 1-3);
3) point sample: centrifugal rear the absorption with liquid-transfering gun checked the grid central authorities that pull on 2 ul;
4) sealing: behind the point sample, the NC film left standstill dry 10 minutes, the skimmed milk power of adding 5% (adding the ratio preparation of 100 ml PBST damping fluids (the 0.01 mol/L PBS that contains 0.05% Tween-20) in 5 g skimmed milk powers) sealing, 37 ℃ left standstill 30 minutes;
5) add primary antibodie: add the monoclonal antibody of tomato yellow leaf curl virus, the skimmed milk power 1:2000 of monoclonal anti body and function 5% doubly dilutes, and hatches 1 hour for 37 ℃;
6) washing: abandon liquid, the NC film shakes wash-out 3 times with PBST, each 3 minutes;
7) add ELIAS secondary antibody: abandon eluent, add the sheep anti-mouse igg two anti-(A3562 Sigma) of alkali phosphatase enzyme mark, the skimmed milk power 1:8000 of two anti-usefulness 5% doubly dilutes, and hatches 1 hour for 37 ℃;
8) washing: abandon liquid, the NC film shakes wash-out 5 times with PBST, each 5 minutes;
9) colour developing: chromogenic substrate NBT 66 ul and BCIP 33 ul(S3771 Promega) be added to mixing in the 10 ml substrate buffer solutions (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5), put into substrate nitrite ion reaction 10 minutes after washed NC film blots with filter paper;
10) cessation reaction: treat that positive control presents purple and abandon the nitrite ion cessation reaction when negative control does not develop the color;
11) record result: ddH
2O soaks the NC film, dries rear record result.
Experimental result as shown in Figure 4, the positive rate of 14 strain tomato samples is the positive contrast of 100%(C7, D7, the negative contrast of C8, D8), show that tomato yellow leaf curl virus is comparatively general in this area, it is comparatively serious to fall ill.
Embodiment two: utilize the method for organizing trace ELISA to detect the situation that infects of tomato yellow leaf curl virus in the tomato plant of field.
Gather tomato 14 strains with disease symptom from the Zhengzhou, henan farmland, again detect according to the method for operating of organizing trace ELISA:
1) NC prepares: rule at the outer paper of NC film with pencil, make the NC film stamp the graticule line vestige, every hole gauge lattice 1 * 1 cm makes marks in the lower right corner of NC film, the smooth double dish central authorities that put into;
2) point sample: use the blade cuts petiole, make otch concordant (Fig. 5), with cut-out section by placing the grid central authorities 3 ~ 5 seconds (Fig. 6) that pull, each sample repeats once (to cut in addition once with same blade, repeat the aforesaid operations step), in the operating process, sample of the every cutting of blade with the alcohol swab wiping once;
3) sealing: behind the point sample, the NC film left standstill dry 10 minutes, add 5% skimmed milk power (adding the ratio preparation of 100 ml PBST damping fluids in 5 g skimmed milk powers) sealing, 37 ℃ left standstill 30 minutes;
4) add primary antibodie: add the monoclonal antibody of tomato yellow leaf curl virus, the skimmed milk power 1:2000 of monoclonal anti body and function 5% doubly dilutes, and hatches 1 hour for 37 ℃;
5) washing: abandon liquid, the NC film contains the 0.01 mol/L PBS of 0.05% Tween-20 with PBST() shake wash-out 3 times, each 3 minutes;
7) add ELIAS secondary antibody: abandon eluent, add the sheep anti-mouse igg two anti-(A3562 Sigma) of alkali phosphatase enzyme mark, the skimmed milk power 1:8000 of two anti-usefulness 5% doubly dilutes, and hatches 1 hour for 37 ℃;
8) washing: abandon liquid, the NC film shakes wash-out 5 times with PBST, each 5 minutes;
9) colour developing: chromogenic substrate NBT 66 ul and BCIP 33 ul(S3771 Promega) be added to (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mixing in the 10 ml substrate buffer solutions, put into substrate nitrite ion reaction 10 minutes after washed film blots with filter paper;
10) cessation reaction: treat that sample presents purple and abandon the nitrite ion cessation reaction when negative control does not develop the color;
11) record result: ddH
2O soaks the NC film, dries rear record result.
Testing result as shown in Figure 7, the sample positive rate is the positive contrast of 100%(D5, D6, the negative contrast of D7, D8), it is consistent to detect acquired results with dot-ELISA.In addition, utilize the Auele Specific Primer PF (5 '-GGCGCGCCCATGTCGAAGCGACCAG-3 ') of tomato yellow leaf curl virus and PR (5 '-GTCGACTTAATTTGATATTGAATCATAGAAATAG-3 ') that this batch sample is carried out the PCR checking, 14 samples all amplify the purpose band (Fig. 8) of about 750 bp, and the PCR experimental data confirms that these 14 field samples all carry tomato yellow leaf curl virus.This shows that two kinds of serology method for quick are accurate, sensitive, reliable, are applicable to applying of field batch samples detection.
Embodiment three: tomato yellow leaf curl virus dot-ELISA and the exploitation of organizing trace ELISA detection kit
1. test principle:
Dot-ELISA and to organize trace ELISA be ELISA detection method take the NC film as solid phase carrier, have easy, quick, special, responsive, be convenient to popularization, do not need the advantage such as specific apparatus, application prospect is wide.The extract point that will contain the tomato yellow leaf curl virus sample to or be impressed on the NC film the dry solid phase antigen that forms; The mouse monoclonal antibody that adds anti-tomato yellow leaf curl virus, then monoclonal antibody and solid phase antigen (tomato yellow leaf curl virus) form antigen-antibody complex; Add the anti-mouse IgG antiantibody (namely two is anti-) of alkali phosphatase enzyme mark, then antiantibody is combined with above-mentioned antigen-antibody complex and is formed Ag-Ab-enzyme mark antiantibody compound again; Add chromogenic substrate, the substrate for enzymatic activity on the compound generates the coloured product of sedimentation type and develops the color.Owing to the step of washing is all arranged between per step, if do not contain in the testing sample tomato yellow leaf curl virus then enzyme labelled antibody will be washed off, substrate does not develop the color and is negative reaction.The quantitative and semi-quantitative that the depth that the visual inspection spot colors has that it's too late is carried out tomato yellow leaf curl virus in the sample detects.
2. main agents and material:
20 * concentrated PBS(0.2 mol/L PBS) 10 ml
TYLCV monoclonal antibody 0.1 ml
AP mark sheep anti-mouse igg two anti-(A3562 Sigma) 0.02 ml
10 * concentrated confining liquid (0.1 mol/L PBST) 10 ml
10 * concentrated antibody dilution (50% skimmed milk power, 0.1mol/L PBST), 20 ml
20 * concentrated cleaning solution (0.2 mol/L PBST), 70 ml
Sealer (skimmed milk power) 15 g
Substrate buffer solution (0.1 mol/L Tris Cl, 0.025 mol/L MgCl, pH9.5) 70 ml
7 of NC films (Amersham)
Above reagent all is stored under 4 ℃
NBT storing solution (S3771 Promega) 0.4 ml
BCIP storing solution (S3771 Promega) 0.2 ml
Above reagent all is stored under-20 ℃
3. operation steps:
Organize trace ELISA step:
1) get tomato stem or petiole, use the blade crosscut, the square section is impressed 3-5 second at film immediately, its Leaf is used the blade crosscut after need tightly being rolled into cylinder.Turn step 4).
The dot-ELISA step:
1) after tomato stem or blade are weighed, press 1:30-1:50(weight: volume, grams per milliliter) adding 0.01 mol/L PBS grinding;
2) centrifugal 3 min of 5000 rpm;
3) get and check on the 2ul on the NC film;
4) drying at room temperature 10 min;
5) the NC film is immersed in room temperature sealing 30-60 min in the confining liquid that contains 5% sealer;
6) the NC film is put into the monoclonal antibody incubated at room 30-60 min that doubly dilutes with antibody diluent 1:2000;
7) cleansing solution is washed film 3-4 time, each 3 min;
8) the NC film is put into the alkali phosphatase enzyme mark sheep anti-mouse igg two anti-incubated at room 30-60 min that doubly dilute with antibody diluent 1:8000;
9) cleansing solution is washed film 5-6 time, each 3 min;
10) chromogenic substrate NBT 66 ul and BCIP 33 ul are added to mixing in the 10 ml substrate buffer solutions, putting into the substrate nitrite ion after washed film blots with filter paper reacts, treat the positive control colour developing obviously, and negative cessation reaction (colour developing 5-20 min) during without any colour developing, namely rinsing is once in tap water, visual inspection result, and record result.
4. damping fluid proportioning:
1) 0.01 mol/L PBS: with deionized water with 20 * concentrated PBS dilutes (namely 1 part 20 * concentrate PBS+19 part deionized water or mineral water) by the 1:19 volume ratio.
2) contain the confining liquid of 5% sealer: with deionized water 10 * concentrated confining liquid is diluted (1 part 10 * concentrated confining liquid+9 parts of deionized waters) by the 1:9 volume ratio, add the 1g sealer by per 20 ml dilution confining liquid after the dilution and be mixed with the confining liquid that contains 5% sealer, the confining liquid for preparing can be preserved one month at 4 ℃ of refrigerators.
3) cleansing solution: with deionized water 20 * concentrated cleaning solution is diluted (or by requirement dilution) (1 part of 20 * concentrated cleaning solution+19 part deionized water) by the 1:19 volume ratio, the cleansing solution after the dilution can be preserved one month at 4 ℃ of environment.
4) antibody diluent: with deionized water 10 * antibody diluent is diluted (1 part of 10 * antibody diluent+9 part deionized water) by the 1:9 volume ratio, add the 1g sealer by per 20 ml dilutions after the dilution and be mixed with antibody diluent.The antibody diluent for preparing can be preserved one month at 4 ℃ of refrigerators.
5) substrate nitrite ion: 66 uL NBT and 33 uL BCIP are added to the substrate buffer solution mixing of 10 mL.
5. points for attention:
1) the NC film is positioned in the middle of 2 protection sheets, not with the direct touch membrane of hand, with tweezers with wear disposable PE gloves and get film;
2) one side of dropping test sample is positive on the NC film, should be up in the whole experimentation;
3) antibody dilutes within 10 min before use;
4) the substrate nitrite ion is now with the current;
5) the positive contrast in nitrocellulose filter CK+ place; The negative contrast in nitrocellulose filter CK-place;
6) the kit temperature of reaction is 4-38 ℃, and optimal reaction temperature is 37 ℃;
7) kit can detect 200 field samples.
6. holding conditions and storage life:
Kit keeps in Dark Place in 2-8 ℃, but antibody-20 ℃ freezing preservation is better, and NBT storing solution and BCIP storing solution must-20 ℃ of freezing preservations.Storage life: this keeping life is 6 months.
Embodiment four: tomato yellow leaf curl virus dot-ELISA and organize trace ELISA detection kit test
Plant detection kit (Fig. 9 and Figure 10) with research and development, respectively by dot-ELISA and organize the method for trace ELISA that the plant sample that the field has disease symptom is detected, the result fast, reliable (Figure 11,12), show that this plant detection kit can use in enormous quantities.
Claims (5)
1. a dot-ELISA rapid identification method that detects the plant infection tomato yellow leaf curl virus is characterized in that comprising the steps:
1) cellulose nitrate (NC) film is prepared: rule at NC film skin paper with pencil, make the NC film stamp the graticule line vestige, every lattice specification 1 * 1 cm makes marks the smooth double dish central authorities that put at NC film one jiao;
2) plant sample is processed: volume ratio 1:30 to 1:50(grams per milliliter by weight) ratio takes by weighing plant stem, and add 0.01 mol/L PBS plant tissue is ground to form the homogenate shape;
3) point sample: draw 2 ul plant supernatants with liquid-transfering gun and put the grid central authorities that pull;
4) sealing: behind the point sample, the NC film left standstill dry 10 minutes, use obtains 5% skimmed milk power sealing in the ratio preparation that 5 g skimmed milk powers add 100 mL PBST damping fluids, and 37 ℃ left standstill 0.5-1 hour, and described PBST damping fluid contains the 0.01 mol/L PBS of 0.05% Tween-20;
5) add primary antibodie: add Chinese patent application and number be the monoclonal antibody of 201210004197.7 tomato yellow leaf curl virus, the skimmed milk power 1:2000 of this monoclonal anti body and function 5% doubly dilutes, and hatches 1 hour for 37 ℃;
6) washing: abandon liquid, the NC film shakes wash-out 3 times with the PBST damping fluid, each 3 minutes;
7) add ELIAS secondary antibody: abandon eluent, the sheep anti-mouse igg two that adds alkali phosphatase enzyme mark is anti-, and the skimmed milk power 1:8000 of two anti-usefulness 5% doubly dilutes, and hatches 1 hour for 37 ℃;
8) washing: abandon liquid, the NC film shakes wash-out 5 ~ 6 times with the PBST damping fluid, each 5 minutes;
9) colour developing: chromogenic substrate NBT 66 ul and BCIP 33 ul are added to mixing in the 10 ml substrate buffer solutions, put into substrate nitrite ion reaction 10 minutes after washed NC film blots with filter paper;
10) cessation reaction: treat that sample on the NC film presents purple and abandon the nitrite ion cessation reaction when negative control does not develop the color;
11) record result: ddH
2O soaks the NC film, dries rear record result.
One kind detect the plant infection tomato yellow leaf curl virus organize trace ELISA method for quick, it is characterized in that comprising the steps:
1) the NC film is prepared: rule at NC film skin paper with pencil, make the NC film stamp the graticule line vestige, every lattice specification 1 * 1 cm makes marks the smooth double dish central authorities that put at NC film one jiao;
2) plant sample is processed and point sample: with blade cuts plant petiole, make otch concordant, by placing the grid central authorities that pull 3 ~ 5 seconds, each plant sample repeats aforesaid operations step once with cut-out section; In the operating process, plant sample of every cutting is changed blade or with the alcohol swab blade of sterilizing;
3) sealing: behind the point sample, the NC film left standstill dry 10 minutes, use the ratio preparation that adds 100 mL PBST damping fluids in 5 g skimmed milk powers to obtain 5% skimmed milk power sealing, 37 ℃ left standstill 0.5-1 hour; Described PBST damping fluid contains the 0.01 mol/L PBS of 0.05% Tween-20;
4) add primary antibodie: add Chinese patent application and number be the monoclonal antibody of 201210004197.7 tomato yellow leaf curl virus, the skimmed milk power 1:2000 of monoclonal anti body and function 5% doubly dilutes, and hatches 1 hour for 37 ℃;
5) washing: abandon liquid, the NC film shakes wash-out 3 times with PBST, each 3 minutes;
7) add ELIAS secondary antibody: abandon eluent, the sheep anti-mouse igg two that adds alkali phosphatase enzyme mark is anti-, and the skimmed milk power 1:8000 of two anti-usefulness 5% doubly dilutes, and hatches 1 hour for 37 ℃;
8) washing: abandon liquid, the NC film shakes wash-out 5 ~ 6 times with the PBST damping fluid, each 5 minutes;
9) colour developing: chromogenic substrate NBT 66 ul and BCIP 33 ul are added to mixing in the 10 ml substrate buffer solutions, put into substrate nitrite ion reaction 10 minutes after washed film blots with filter paper;
10) cessation reaction: treat that sample on the NC film presents purple and abandon the nitrite ion cessation reaction when negative control does not develop the color;
11) record result: ddH
2O soaks the NC film, dries rear record result.
3. a tomato yellow leaf curl virus dot-ELISA and organize trace ELISA detection kit is characterized in that comprising the steps:
1) kit reagent and material:
20
*Concentrated PBS(0.2 mol/L) 10 ml
Monoclonal antibody 0.1 ml
AP mark sheep anti-mouse igg two anti-0.02 ml
10
*Concentrated confining liquid 10 ml
10
*Concentrated antibody dilution 20 ml
20
*Concentrated cleaning solution 70 ml
Sealer 15 g
Substrate buffer solution 70 ml
7 of NC films
NBT storing solution 0.4 ml
BCIP storing solution 0.2 ml
2) kit operation steps:
Organize trace ELISA step:
2.1) get tomato stem or petiole, use the blade crosscut, the square section is impressed 3-5 second at film immediately; Turn step 2.4;
The dot-ELISA step:
2.1) after the tomato stem weighs, volume ratio 1:30-1:50(grams per milliliter by weight) add 0.01 mol/L PBS and grind;
2.2) centrifugal 3 minutes of 5000 rpm;
2.3) get and check on the NC film on 2 ul;
2.4) drying at room temperature 10 minutes;
2.5) the NC film is immersed in and contains 1 of 5% sealer
*The room temperature sealing is 30-60 minute in the confining liquid;
2.6) the NC film puts into 1
*In the monoclonal antibody that antibody diluent 1:2000 doubly dilutes incubated at room 30-60 minute;
2.7) 1
*Cleansing solution is washed film 3-4 time, each 3 minutes;
2.8) the NC film puts into 1
*The alkali phosphatase enzyme mark sheep anti-mouse igg two that antibody diluent 1:8000 doubly dilutes resists middle incubated at room 30-60 minute;
2.9) 1
*Cleansing solution is washed film 5-6 time, each 3 minutes;
2.10) chromogenic substrate NBT 66 ul and BCIP 33 ul are added to mixing in the 10 ml substrate buffer solutions, putting into the substrate nitrite ion after washed film blots with filter paper reacts, treat the positive control colour developing obviously, and negative cessation reaction during without any colour developing, developing time 5-20 minute, namely in tap water rinsing once, visual inspection is also recorded the result;
3) kit application notice:
3.1) the NC film is positioned in the middle of 2 protection sheets, directly do not touch the NC film with hand;
3.2) one side that drips test sample on the NC film be the front, should be up in the whole experimentation;
3.3) dilution in the antibody 10 minutes before use;
3.4) the substrate nitrite ion is now with the current;
3.5) the positive contrast in NC film CK+ place; The negative contrast in NC film CK-place;
3.6) the kit optimal reaction temperature is 37 ℃;
3.7) kit can detect 200 field samples;
4) holding conditions and storage life:
Kit keeps in Dark Place in 2-8 ℃, but wherein antibody-20 ℃ freezing or 2-8 ℃ of preservation, and NBT storing solution and BCIP storing solution must-20 ℃ of freezing preservations; Storage life: this kit term of validity is 6 months.
4. application such as claim 1,2,3 each described methods or kit, it is characterized in that described method is applicable to the host of tomato yellow leaf curl virus---plant of Solanaceae, described plant of Solanaceae comprises tomato, capsicum, eggplant, tobacco, black nightshade, datura, and sampling point is stem or the petiole of plant.
5. application such as claim 1,2,3 each described methods or kit, it is characterized in that described method can investigate the band of a large amount of samples in field poison situation, calculate tomato yellow leaf curl virus at the infection rate in field, the field occurrence and distribution of assessment tomato yellow leaf curl and popular outburst trend are in order in time take prophylactico-therapeutic measures.
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| CN2012104262273A CN102928598A (en) | 2012-10-30 | 2012-10-30 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012104262273A CN102928598A (en) | 2012-10-30 | 2012-10-30 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
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| CN2012104262273A Pending CN102928598A (en) | 2012-10-30 | 2012-10-30 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
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| CN103563897A (en) * | 2013-07-12 | 2014-02-12 | 江苏省农业科学院 | Method for reducing plant virus carrying rate in insect medium by utilizing gossypol |
| CN104198713A (en) * | 2014-09-05 | 2014-12-10 | 云南省农业科学院生物技术与种质资源研究所 | Method for rapidly detecting tomato spotted wilf virus in thrips |
| CN104515853A (en) * | 2014-11-10 | 2015-04-15 | 浙江大学 | Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application |
| CN105548543A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Kit for detecting yellow spotting virus of peanuts, as well as detection method and application thereof |
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| CN109929812A (en) * | 2019-02-15 | 2019-06-25 | 云南省农业科学院生物技术与种质资源研究所 | Marmor solani strain and its construction method and application in a kind of Potato Cultivars cooperation 88 |
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| CN103563897A (en) * | 2013-07-12 | 2014-02-12 | 江苏省农业科学院 | Method for reducing plant virus carrying rate in insect medium by utilizing gossypol |
| CN104198713A (en) * | 2014-09-05 | 2014-12-10 | 云南省农业科学院生物技术与种质资源研究所 | Method for rapidly detecting tomato spotted wilf virus in thrips |
| CN104198713B (en) * | 2014-09-05 | 2016-02-10 | 云南省农业科学院生物技术与种质资源研究所 | A kind of method of tomato spotted wilf virus in quick detection thrips body |
| CN104515853A (en) * | 2014-11-10 | 2015-04-15 | 浙江大学 | Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application |
| CN105548543A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Kit for detecting yellow spotting virus of peanuts, as well as detection method and application thereof |
| CN105548544A (en) * | 2016-02-04 | 2016-05-04 | 云南省农业科学院生物技术与种质资源研究所 | Detection kit of hippeastrum chlorotic ringspot viruses (HCRV) as well as detection method and application of detection kit |
| CN106226524A (en) * | 2016-07-07 | 2016-12-14 | 福建省农业科学院食用菌研究所 | A kind of detection method of edible fungi dsRNA virus |
| CN106226524B (en) * | 2016-07-07 | 2018-10-09 | 福建省农业科学院食用菌研究所 | A kind of detection method of edible mushroom dsRNA viruses |
| CN109929811A (en) * | 2019-02-15 | 2019-06-25 | 云南省农业科学院生物技术与种质资源研究所 | The red middle potato virus X strain in pasture and its construction method and application in a kind of Potato Cultivars |
| CN109929812A (en) * | 2019-02-15 | 2019-06-25 | 云南省农业科学院生物技术与种质资源研究所 | Marmor solani strain and its construction method and application in a kind of Potato Cultivars cooperation 88 |
| CN111929444A (en) * | 2020-08-12 | 2020-11-13 | 四川大学华西医院 | Effective immunoblotting PVDF membrane labeling method, primary anti-incubation method and elution and color development method |
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Application publication date: 20130213 |