CN104515853A - Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application - Google Patents
Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application Download PDFInfo
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- CN104515853A CN104515853A CN201410627387.3A CN201410627387A CN104515853A CN 104515853 A CN104515853 A CN 104515853A CN 201410627387 A CN201410627387 A CN 201410627387A CN 104515853 A CN104515853 A CN 104515853A
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Abstract
The invention discloses a serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application A hybridoma cell strain 1A5By capable of secreting monoclonal antibody against tomato spotted wilt virus and having a preservation number of CGMCC No.9343 is utilized to establish a dot-ELISA method for detection of tomato spotted wilt virus carried by individual thrips, to research and develop a rapid detection kit, and to rapidly detect the virus carried rate of the field thrips carrying tomato spotted wilt virus. The invention can be used in mass rapid investigation of virus carried rate of the thrips and in early prediction of tendency and outbreak of the tomato spotted wilt virus. The detection method is fast, efficient, sensitive and specific, and has high throughput and easy operation, and provides technical support for the rapid diagnosis, prediction, forecast and scientific prevention and control of the tomato spotted wilt virus disease.
Description
Technical field
Field belonging to the present invention is biological technical field, especially relates to a kind of method for quick and application of carrying the thrips of tomato spotted wilf virus.
Background technology
1919, tomato spotted wilf virus (Tomato spotted wilt virus, TSWV) on the tomato of Australia, find all have generation in global tropical, subtropics, Temperate Region in China at present first, be distributed widely in multiple countries such as Europe, North America, South America, asia and ocenia.TSWV host range is very extensive, can encroach on 160 kinds of dicotyledons and 10 kinds of monocotyledons, cause serious economic loss to agricultural production.TSWV causes disease plant blade to form little blackspot, leaf back along the pulse in purple, the downright bad streak of brown, diseased plant downgrade or fallen leaves in wilting shape, immature fruit there is chlorisis ring spot, brown necrotic plaque, ripening fruits wheel line obviously, mummy.Not yet bring out the resistant variety of anti-TSWV at present, TSWV disease control is very difficult.TSWV is Europe and Mediterranean Plant Protection Organization (EPPO) A2 class quarantine harmful organisms, and Ye Shi China enters the territory plant quarantine harmful organism.
TSWV is a kind of RNA virus, and route of transmission is wide, and virus can juice contact transmission, kind biography and amboceptor thrips biography poison.Amboceptor thrips with persistent fashion pass poison, be TSWV main biography poison mode, pass malicious thrips comprise onion thrips (
thrips tabaci), beans thrips (
t.Setosus), thrips (
frankliniella schutzei), tobacco brown thrip (
f.Fusca) and alfalfa thrips (
f.Occidentatis) etc.Thrips can only obtain virus in larval phase, and passing poison needs to breed in vivo, and onion thrips is the shortest, and to obtain the poison phase be 15 ~ 30 minutes, and beans thrips needs 30 minutes, and the time, long Virus spreading rate raised, and thrips is once band poison, and pass poison and reach 20 days, tool passes malicious ability throughout one's life.Virus inoculation is many to be obtained when the shallow epidermis of tomato leaf table cell is sucked, and generally namely falls ill through 4 days incobations.The exosper such as tomato, cineraria band poison, does not enter embryo.The main cause causing TSWV popular with malicious thrips insect density.
Research in the past for TSWV concentrates on susceptible plant tissue, and seldom research passes virus mediator thrips.When observing in plant Deng people or TSWV detected, often TSWV gets up at Field epidemic, and at this moment prevention and control are late.If at crop premorbid, the band poison amount that a kind of way can be had to detect fast pass virus mediator thrips, thus on Accurate Prediction crop disease a situation arises, for taking prevention and control measure to strive for Best Times in time, be the preferred approach of control TSWV happening and prevelence.Fresh rare report research thrips is with malicious situation in the whole world, its method can only rely on RT-PCR,---reverse transcription---these steps of pcr amplification---electrophoretic analysis---genome sequence determination that RT-PCR needs to extract through geneome RNA, not only loaded down with trivial details, the consuming time length of experimental implementation process, experimentation cost are higher, be unfavorable for the extensive detection of field Virus Sample, layman has also been difficult to; And thrips is very little, body is about 1 millimeter, wants single head thrips RT-PCR and detects very difficult.And serological method is applicable to field sample batch detection, layman also can easily grasp.But also do not report the serological method detecting TSWV in thrips body at present.
The present invention utilizes tomato spotted wilf virus monoclonal antibody, set up and a kind ofly detect dot enzyme-linked immuno absorption method (the dot enzyme-linked immunosorbent assay that single head thrips carries tomato spotted wilf virus, and research and develop quick detection kit dot-ELISA).This patent of invention can be applicable to the extensive fast investigation of band poison rate of field thrips, the happening and prevelence outburst trend of look-ahead tomato spotted wilf virus disease.This detection method rapidly and efficiently, sensitive special, high flux is easy and simple to handle again, for tomato spotted wilf virus disease quick diagnosis, early prediction forecast and science bridle technical support is provided.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of quick detection single head thrips to carry serological method and the application thereof of tomato spotted wilf virus.
Quick detection single head thrips carries the step of the serological method of tomato spotted wilf virus:
1) collection of field thrips, grinding;
2) draw thrips homogenate 2 μ L, point sample, in nitrocellulose filter, leaves standstill and dries 10 minutes;
3) 37 DEG C of closed 30-60 minute in the PBST of 5% skimmed milk power;
4) above-mentioned nitrocellulose filter puts into the preserving number that doubly dilutes of skimmed milk power 1:3000 with 5% is the monoclonal antibody of the anti-tomato spotted wilf virus of CGMCC No.9343 hybridoma secretion, hatch 1-2 hour for 37 DEG C, hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 3rd, 2014, and Classification And Nomenclature is: secrete anti-tomato spotted wilf virus (TSMV) monoclonal antibody hybridoma;
5) nitrocellulose filter PBST damping fluid shakes wash-out 3 times, each 3 minutes, abandons eluent;
6) nitrocellulose filter puts into the Sigma company article No. that doubly dilutes of skimmed milk power 1:2000 with 5% is that the sheep anti-mouse igg two of the horseradish peroxidase-labeled of A4416 resists, and hatches 1-2 hour for 37 DEG C;
7) nitrocellulose filter PBST damping fluid shakes wash-out 5 times, and each 3-5 minute, abandons eluent;
8) after nitrocellulose filter dries, the TMB nitrite ion that Promega company article No. is W4121 is put into, lucifuge colour developing 5-15 minute;
9) when until positive presents blueness, negative control does not develop the color, record result.
The collection of field thrips, the step of grinding:
1) thrips gathers: gather the plant tissues such as the intensive flower of thrips, leaf from large Tanaka;
2) thrips grinding: dip single head thrips with writing brush, be placed on clean sealed membrane, every thrips drips 2 μ L 0.05 mol/L PBS damping fluids, with being milled to till naked eyes can not see and obviously organizing at the bottom of 0.5 mL centrifuge tube.
Quick detection thrips carries the kit of tomato spotted wilf virus, comprises following reagent, material and operation steps:
1) main agents and material in kit:
0.05 mol/L PBS 10 ml
TSWV monoclonal antibody 0.1 ml
Positive control 0.1 ml
Negative control 0.1 ml
HRP marks anti-0.1 ml of sheep anti mouse two
TMB assay chromogenic substrate solution 10 ml
10 × concentrated confining liquid 10 ml
10 × concentrated antibody dilution 20 ml
20 × concentrated cleaning solution 60 ml
Sealer 15 g
Nitrocellulose filter 5
2) kit operation steps:
2.1) single head thrips is placed on sealed membrane, adds 2 ul 0.05 mol/L PBS, grinds with at the bottom of 0.5 ml centrifuge tube;
2.2) 2 ul homogenate points are got on nitrocellulose filter and NC film, 37 DEG C of dryings 10 minutes;
2.3) during NC film is immersed in containing 5% sealer 1 × confining liquid, room temperature closes 30 minutes;
2.4) NC film is put into the monoclonal antibody 37 DEG C doubly diluted with 1 × antibody diluent 1:3000 and is hatched 30-60 minute;
2.5) 1 × cleansing solution washes film 3 times, each 3 minutes;
2.6) NC film put into the HRP that doubly dilutes with 1 × antibody diluent 1:2000 mark sheep anti-mouse igg two anti-37 DEG C hatch 30-60 minute;
2.7) 1 × cleansing solution washes film 5 times, each 3 minutes;
2.8) after blotting film with filter paper, tmb substrate nitrite ion is added drop-wise to film surface and carries out chromogenic reaction, visual results, treat positive control colour developing obviously, and feminine gender is without any record testing result during colour developing, developing time is about 5-15 minute.
Detect single head thrips and carry the serology rapid identification method of tomato spotted wilf virus and the application of kit thereof, be to utilize the method and the band poison situation of detection kit to field thrips thereof to investigate, accurate Calculation thrips is with malicious rate, for prediction tomato spotted wilf virus in field happening and prevelence outburst trend and science bridle technical support is provided.
The beneficial effect that the present invention compared with prior art has:
1) the invention provides one specially for biography virus mediator thrips, detect the serological method carrying TSWV virus in its body fast.The method is highly sensitive, high specificity, easy and simple to handle, and rapidly and efficiently, testing result is reliable;
2) present invention also offers and a kind ofly detect the quick detection kit that thrips carries tomato spotted wilf virus.This kit does not rely on instrument, cost is little, easy and simple to handle; Can detect hundreds of to several thousand thrips samples, high flux is time-saving and efficiency again, is suitable for layman at field large-scale promotion application simultaneously;
3) the present invention can realize the detection of single head thrips being carried to TSWV, and degree of accuracy is high;
4) utilize the present invention can carry out large-scale inquiry to the band poison situation of field thrips, accurate Calculation thrips is with malicious rate, and the sick popular outburst trend in field of early prediction tomato spotted wilf virus is to take prophylactico-therapeutic measures in time.
Accompanying drawing explanation
Thrips sample belt poison rate testing result is raised in Fig. 1 part Experiment room;
Fig. 2 detects the kit that thrips carries tomato spotted wilf virus;
Fig. 3 application kit detects the band poison situation of field thrips.
Embodiment
Quick detection single head thrips carries the step of the serological method of tomato spotted wilf virus:
1) collection of field thrips, grinding;
2) draw thrips homogenate 2 μ L, point sample, in nitrocellulose filter, leaves standstill and dries 10 minutes;
3) 37 DEG C of closed 30-60 minute in the PBST of 5% skimmed milk power;
4) above-mentioned nitrocellulose filter puts into the preserving number that doubly dilutes of skimmed milk power 1:3000 with 5% is the monoclonal antibody of the anti-tomato spotted wilf virus of CGMCC No.9343 hybridoma secretion, hatches 1-2 hour for 37 DEG C;
5) nitrocellulose filter PBST damping fluid shakes wash-out 3 times, each 3 minutes, abandons eluent;
6) nitrocellulose filter puts into the Sigma company article No. that doubly dilutes of skimmed milk power 1:2000 with 5% is that the sheep anti-mouse igg two of the horseradish peroxidase-labeled of A4416 resists, and hatches 1-2 hour for 37 DEG C;
7) nitrocellulose filter PBST damping fluid shakes wash-out 5 times, and each 3-5 minute, abandons eluent;
8) after nitrocellulose filter dries, the TMB nitrite ion that Promega company article No. is W4121 is put into, lucifuge colour developing 5-15 minute;
9) when until positive presents blueness, negative control does not develop the color, record result.
The collection of field thrips, the step of grinding:
1) thrips gathers: gather the plant tissues such as the intensive flower of thrips, leaf from large Tanaka;
2) thrips grinding: dip single head thrips with writing brush, be placed on clean sealed membrane, every thrips drips 2 μ L 0.05 mol/L PBS damping fluids, with being milled to till naked eyes can not see and obviously organizing at the bottom of 0.5 mL centrifuge tube.
Secrete the hybridoma cell strain 1A5 of anti-tomato spotted wilf virus monoclonal antibody, it can secrete the monoclonal antibody specific of anti-tomato spotted wilf virus, hybridoma cell strain 1A5 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 3rd, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, preserving number is CGMCC No.9343;
Quick detection thrips carries a kit for tomato spotted wilf virus, comprises following reagent, material and operation steps:
1) main agents and material in kit:
0.05 mol/L PBS 10 ml
TSWV monoclonal antibody 0.1 ml
Positive control 0.1 ml
Negative control 0.1 ml
HRP marks anti-0.1 ml of sheep anti mouse two
TMB assay chromogenic substrate solution 10 ml
10 × concentrated confining liquid 10 ml
10 × concentrated antibody dilution 20 ml
20 × concentrated cleaning solution 60 ml
Sealer 15 g
Nitrocellulose filter 5
2) kit operation steps:
2.1) single head thrips is placed on sealed membrane, adds 2 ul 0.05 mol/L PBS, grinds with at the bottom of 0.5 ml centrifuge tube;
2.2) 2 ul homogenate points are got on nitrocellulose filter and NC film, 37 DEG C of dryings 10 minutes;
2.3) during NC film is immersed in containing 5% sealer 1 × confining liquid, room temperature closes 30 minutes;
2.4) NC film is put into the monoclonal antibody 37 DEG C doubly diluted with 1 × antibody diluent 1:3000 and is hatched 30-60 minute;
2.5) 1 × cleansing solution washes film 3 times, each 3 minutes;
2.6) NC film put into the HRP that doubly dilutes with 1 × antibody diluent 1:2000 mark sheep anti-mouse igg two anti-37 DEG C hatch 30-60 minute;
2.7) 1 × cleansing solution washes film 5 times, each 3 minutes;
2.8) after blotting film with filter paper, tmb substrate nitrite ion is added drop-wise to film surface and carries out chromogenic reaction, visual results, treat positive control colour developing obviously, and feminine gender is without any record testing result during colour developing, developing time is about 5-15 minute.
Detect single head thrips and carry the serology rapid identification method of tomato spotted wilf virus and the application of kit thereof, be to utilize the method and the band poison situation of detection kit to field thrips thereof to investigate, accurate Calculation thrips is with malicious rate, for prediction tomato spotted wilf virus in field happening and prevelence outburst trend and science bridle technical support is provided.
Below by way of drawings and Examples, the invention will be further elaborated.
Embodiment one: thrips sample belt poison rate detects
Gather laboratory rearing thrips 160, dip single head thrips, be placed on clean sealed membrane with writing brush, every drips 2 ul 0.05 mol/L PBS, grinds with at the bottom of 0.5 ml centrifuge tube.Draw 1-2 μ L thrips homogenate with liquid-transfering gun, put in nitrocellulose filter central authorities, and the positive, negative control are set.Film is left standstill and dries, add the skimmed milk power of about 10 ml 5%, close 30 minutes for 37 DEG C; Abandon skimmed milk power, add the monoclonal antibody of the tomato spotted wilf virus that 1:3000 doubly dilutes, 37 DEG C of stationary incubation 1 hour; Abandon liquid, with the 0.01 mol/L PBS of PBST(containing 0.05% Tween-20) shake wash-out 3 times, each 3 minutes; Abandon eluent, add two anti-(the A4416 Sigma) that horseradish peroxidase (HRP) that 1:2000 doubly dilutes marks, 37 DEG C of stationary incubation 1 hour; Abandon liquid, on horizontal shaker, shake wash-out 5 times with PBST, each 5 minutes; Abandon eluent, add TMB nitrite ion (W4121 Promega) colour developing after being blotted by film filter paper, lucifuge leaves standstill 5 ~ 15 minutes, abandons nitrite ion cessation reaction, Taking Pictures recording result when feminine gender does not develop the color until the positive presents blueness; Calculate band poison rate (being with malicious rate=number positive/total number of samples × 100%) of thrips.Testing result shows in 160 thrips to be had 38 be positive (Fig. 1), and as calculated, the band poison rate of thrips to TSWV is 23.75%, and this thrips can be used for the biography poison experiment of healthy tomato.
Embodiment two: detect the exploitation that thrips carries tomato spotted wilf virus dot-ELISA kit
1. detect thrips to carry tomato spotted wilf virus (TSWV) dot-ELISA kit (Fig. 2) and comprise following material and reagent:
1) kit operation instructions 1 part
2) reagent and material:
0.05 mol/L PBS 10 ml
TSWV monoclonal antibody 0.1 ml
Positive control (being with malicious thrips homogenate) 0.1 ml
Negative control (non-band poison thrips homogenate) 0.1 ml
HRP marks anti-0.1 ml of sheep anti mouse two
TMB assay chromogenic substrate solution 10 ml
10 × concentrated confining liquid 10 ml
10 × concentrated antibody dilution 20 ml
20 × concentrated cleaning solution 60 ml
Sealer 15 g
NC film 5
2. kit operation instructions comprise:
2.1 kit principles:
The ELISA detection method that dot-ELISA is is solid phase carrier with nitrocellulose filter (NC film), have easy, quick, special, responsive, be convenient to promote, do not need the advantages such as specific apparatus, application prospect is wide.Carry the homogenate point of the thrips sample of TSWV on NC film, dry formation solid phase antigen; Add the mouse monoclonal antibody of anti-TSWV, then monoclonal antibody and solid phase antigen TSWV form antigen-antibody complex; Add the against murine IgG antiantibody (namely two resist) that horseradish peroxidase (HRP) marks again, then antiantibody is combined with above-mentioned antigen-antibody complex and forms Ag-Ab-enzyme mark antiantibody compound; Add chromogenic substrate, the substrate for enzymatic activity on compound generates sedimentation type color products and develops the color.Owing to often all there being the step of washing between step, if not containing TSWV in thrips sample to be measured, then enzyme labelled antibody will be washed off, and substrate does not develop the color and the reaction that is negative.The depth that visual inspection spot colors has that it's too late detects to the quantitative and semi-quantitative carrying out TSWV in thrips.
2.2 operation stepss:
1) single head thrips is placed on sealed membrane, adds 2 ul 0.05 mol/L PBS, grinds with at the bottom of 0.5 ml centrifuge tube;
2) 2 ul homogenate points are got on cellulose nitrate (NC) film, drying at room temperature 10 min;
3) during NC film is immersed in containing 5% sealer 1 × confining liquid, room temperature closes 30 min;
4) NC film puts into the monoclonal antibody incubated at room 30-60 min doubly diluted with 1 × antibody diluent 1:3000;
5) film is washed 3-4 time with 1 × cleansing solution, each 3 min;
6) NC film is put into the HRP doubly diluted with 1 × antibody diluent 1:2000 and is marked the anti-incubated at room 30-60 min of sheep anti-mouse igg two;
7) film is washed 5-6 time with 1 × cleansing solution, each 5 min;
8) after blotting film with filter paper, tmb substrate nitrite ion is added drop-wise to film surface and carries out chromogenic reaction, visual results, treat positive control colour developing obviously, and feminine gender is without any record testing result during colour developing, developing time is about 5-15 min.
2.3 buffer formulation:
1) phosphate buffer (PBS, 0.05 mol/L, pH7.4).
2) containing the confining liquid of 5% sealer: with deionized water, 10 × concentrated confining liquid is undertaken diluting (1 part 10 × concentrated confining liquid+9 parts of deionized waters) by 1:9 volume ratio, dilute confining liquids by every 20 ml after dilution to add 1g sealer and be mixed with confining liquid containing 5% sealer, the confining liquid prepared can preserve one month at 4 DEG C of refrigerators.
3) cleansing solution: undertaken diluting (or by requirement dilution) (1 part of 20 × concentrated cleaning solution+19 parts of deionized waters) by 1:19 volume ratio by 20 × concentrated cleaning solution with deionized water, the cleansing solution after dilution can preserve month at 4 DEG C of environment.
4) antibody diluent: with deionized water, 10 × antibody diluent is undertaken diluting (1 part of 10 × antibody diluent+9 parts of deionized waters) by 1:9 volume ratio, add 1g sealer by every 20 ml dilutions after dilution and be mixed with antibody diluent.The antibody diluent prepared can preserve one month at 4 DEG C of refrigerators.
2.4 points for attention:
1) nitrocellulose filter is positioned in the middle of 2 protection sheets, not with the direct touch membrane of hand, with tweezers or wear disposable PE gloves and get film;
2) NC film dripping the one side detecting sample is front, should be upward in whole experimentation;
3) antibody dilutes within 10 min before use;
4) NC film CK+ place is positive control; NC film CK-place is negative control (the thrips homogenate that point is healthy);
5) kit temperature of reaction is 4-38 DEG C, and optimal reaction temperature is 37 DEG C;
6) kit can detect 200 thrips.
2.5 holding conditions and storage life:
Kit keeps in Dark Place in 2-8 DEG C, and antibody-20 DEG C of freezen protective are better.This keeping life is 6 months.
Embodiment three: utilize kit to detect field thrips and be with malicious rate
Detect the band poison rate of field thrips sample with this kit, can detect hundreds of head thrips simultaneously, result fast, reliable, high flux is easy and simple to handle again, for the happening and prevelence of this disease of early prediction does technical support.Fig. 3 is the partial results utilizing this kit to detect land for growing field crops, Yunnan thrips.Testing result shows in 258 thrips to be had 26 be positive (Fig. 3), and as calculated, the band poison rate of thrips to TSWV is 10.08%, predicts that will there be TSWV happening and prevelence trend on this ground, suggestion pesticide control thrips, the popular and outburst of prevention TSWV.
Claims (4)
1. detect the serological method that single head thrips carries tomato spotted wilf virus fast, it is characterized in that comprising the steps:
1) collection of field thrips, grinding;
2) draw thrips homogenate 2 μ L, point sample, in nitrocellulose filter, leaves standstill and dries 10 minutes;
3) 37 DEG C of closed 30-60 minute in the PBST of 5% skimmed milk power;
4) above-mentioned nitrocellulose filter puts into the preserving number that doubly dilutes of skimmed milk power 1:3000 with 5% is the monoclonal antibody of the anti-tomato spotted wilf virus of CGMCC No.9343 hybridoma secretion, hatches 1-2 hour for 37 DEG C;
5) nitrocellulose filter PBST damping fluid shakes wash-out 3 times, each 3 minutes, abandons eluent;
6) nitrocellulose filter puts into the Sigma company article No. that doubly dilutes of skimmed milk power 1:2000 with 5% is that the sheep anti-mouse igg two of the horseradish peroxidase-labeled of A4416 resists, and hatches 1-2 hour for 37 DEG C;
7) nitrocellulose filter PBST damping fluid shakes wash-out 5 times, and each 3-5 minute, abandons eluent;
8) after nitrocellulose filter dries, the TMB nitrite ion that Promega company article No. is W4121 is put into, lucifuge colour developing 5-15 minute;
9) when until positive presents blueness, negative control does not develop the color, record result.
2. the method for claim 1, it is characterized in that the collection of described field thrips, grinding comprise the steps:
1) thrips gathers: gather the plant tissues such as the intensive flower of thrips, leaf from large Tanaka;
2) thrips grinding: dip single head thrips with writing brush, be placed on clean sealed membrane, every thrips drips 2 μ L 0.05 mol/L PBS damping fluids, with being milled to till naked eyes can not see and obviously organizing at the bottom of 0.5 mL centrifuge tube.
3. detect the kit that thrips carries tomato spotted wilf virus fast, it is characterized in that comprising following reagent, material and operation steps:
1) main agents and material in kit:
0.05 mol/L PBS 10 ml
TSWV monoclonal antibody 0.1 ml
Positive control 0.1 ml
Negative control 0.1 ml
HRP marks anti-0.1 ml of sheep anti mouse two
TMB assay chromogenic substrate solution 10 ml
10 × concentrated confining liquid 10 ml
10 × concentrated antibody dilution 20 ml
20 × concentrated cleaning solution 60 ml
Sealer 15 g
Nitrocellulose filter 5
2) kit operation steps:
2.1) single head thrips is placed on sealed membrane, adds 2 ul 0.05 mol/L PBS, grinds with at the bottom of 0.5 ml centrifuge tube;
2.2) 2 ul homogenate points are got on nitrocellulose filter and NC film, 37 DEG C of dryings 10 minutes;
2.3) during NC film is immersed in containing 5% sealer 1 × confining liquid, room temperature closes 30 minutes;
2.4) NC film is put into the monoclonal antibody 37 DEG C doubly diluted with 1 × antibody diluent 1:3000 and is hatched 30-60 minute;
2.5) 1 × cleansing solution washes film 3 times, each 3 minutes;
2.6) NC film put into the HRP that doubly dilutes with 1 × antibody diluent 1:2000 mark sheep anti mouse two anti-37 DEG C hatch 30-60 minute;
2.7) 1 × cleansing solution washes film 5 times, each 3 minutes;
2.8) after blotting film with filter paper, tmb substrate nitrite ion is added drop-wise to film surface and carries out chromogenic reaction, visual results, treat positive control colour developing obviously, and feminine gender is without any record testing result during colour developing, developing time is about 5-15 minute.
4. the detection single head thrips as described in any one of claim 1-3 carries the serology rapid identification method of tomato spotted wilf virus and the application of kit thereof, it is characterized in that utilizing the method and the band poison situation of detection kit to field thrips thereof to investigate, accurate Calculation thrips is with malicious rate, for prediction tomato spotted wilf virus in field happening and prevelence outburst trend and science bridle technical support is provided.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410627387.3A CN104515853B (en) | 2014-11-10 | 2014-11-10 | A kind of quick detection single head thrips carries serological method and the application thereof of tomato spotted wilf virus |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410627387.3A CN104515853B (en) | 2014-11-10 | 2014-11-10 | A kind of quick detection single head thrips carries serological method and the application thereof of tomato spotted wilf virus |
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| Publication Number | Publication Date |
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| CN104515853A true CN104515853A (en) | 2015-04-15 |
| CN104515853B CN104515853B (en) | 2016-04-20 |
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| CN114794120A (en) * | 2022-05-25 | 2022-07-29 | 中国农业科学院烟草研究所 | Method for reducing toxic amount of TSWV in thrips adults |
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| WO2003104449A2 (en) * | 2002-06-11 | 2003-12-18 | Phytovation Bv | Improvements in or relating to protein production |
| CN102928598A (en) * | 2012-10-30 | 2013-02-13 | 浙江大学 | 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 |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN103757132A (en) * | 2013-12-25 | 2014-04-30 | 中国农业科学院蔬菜花卉研究所 | Kit for detecting tomato spotted wilf virus infection and application thereof |
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| WO2003104449A2 (en) * | 2002-06-11 | 2003-12-18 | Phytovation Bv | Improvements in or relating to protein production |
| CN102928598A (en) * | 2012-10-30 | 2013-02-13 | 浙江大学 | 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 |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN103757132A (en) * | 2013-12-25 | 2014-04-30 | 中国农业科学院蔬菜花卉研究所 | Kit for detecting tomato spotted wilf virus infection and application thereof |
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Cited By (2)
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| CN114794120A (en) * | 2022-05-25 | 2022-07-29 | 中国农业科学院烟草研究所 | Method for reducing toxic amount of TSWV in thrips adults |
| CN114794120B (en) * | 2022-05-25 | 2023-08-11 | 中国农业科学院烟草研究所 | Method for reducing TSWV (total suspended solid Virus) toxin carrying amount in adult thrips |
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