CN101430335A - Immunity detection reagent kit for rice black-streaked dwarf fijivirus in single-head small brown rice planthopper - Google Patents
Immunity detection reagent kit for rice black-streaked dwarf fijivirus in single-head small brown rice planthopper Download PDFInfo
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- CN101430335A CN101430335A CNA200810235031XA CN200810235031A CN101430335A CN 101430335 A CN101430335 A CN 101430335A CN A200810235031X A CNA200810235031X A CN A200810235031XA CN 200810235031 A CN200810235031 A CN 200810235031A CN 101430335 A CN101430335 A CN 101430335A
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Abstract
The invention discloses an immunodetection kit specially used for rice black-streaked dwarf virus in single planthopper and a using method thereof. Specific polyclonal antibodies of the rice black-streaked dwarf virus (RBSDV) are utilized to build a dot immuno-binding assay (DIBA) detecting method which is used for building the detection kit for rice black-streaked dwarf virus, main reagents of the kit are working solution or concentrated solution, and the kit has the characteristics of convenient use, simple operation, and the like. And simultaneously, the use of the rice black-streaked dwarf virus detection kit can be a standard program or a reduced program, which can greatly broaden the using ranges and applicable persons of the kit and facilitate the population and application of the technique to establishment units, and further, the kit is applied in the fields of plant disease prognosis and prediction and the formulation of prevention and curing strategies, and the like. In addition, the kit can be used for measuring the inoculation strength in varietal resistance seed selection of rice black-streaked dwarf virus, and the like.
Description
Technical field
The present invention relates to a kind of the be exclusively used in immunity detection reagent of rice black-streaked dwarf virus in the single head small brown rice planthopper and the application on agricultural thereof, belong to the agricultural cience and farming techniques field.
Background technology
(Rice black-streaked dwarf virus is a kind of virus disease of being propagated by small brown rice planthopper RBSDV) to rice black-streaked dwarf virus, infects paddy rice and causes black streaked dwarf virus of rice, and paddy rice is in a single day susceptible just not to have results.This disease from 1963 since Yuyao county, China Zhejiang Province finds, mainly endanger in long-grained nonglutinous rice producing region, south, Zhejiang last century, and rarely have discovery in japonica rice main producing regions such as Jiangsu.Stripe disease is caused disaster in provinces such as Jiangsu outbursts at the beginning of 2004, and the rapid large scale applications of a collection of disease-resistant variety such as town rice 88, Xu rice No. 3 and salt rice No. 8 have been played crucial effect to the anti-stool control mode of the integral body of disease.Further studies show that, this class stripe disease disease-resistant variety is generally relatively poor to the resistance of RBSDV, since the MRDV that same cause of disease causes also be in recent years the popular phase for providing of disease sufficient malicious source, the increase year after year of small brown rice planthopper generating capacity in addition, plant protection scholarly forecast black streaked dwarf virus of rice have the potential risk that popular harm takes place.Really the RSV disease-resistant variety promotes that black streaked dwarf virus of rice just takes place popular after 2 years in japonica rice main producing regions such as Jiangsu,, the Huaibei coastal in this province by disease in 2007 according to Jiangsu Province's plant protection unit statistics and in the ground such as Yancheng, Lianyun Harbour, Taizhou, Huaian, Yangzhou and Xuzhou of going to river seriously take place, the area that harm takes place then reaches 30.8 ten thousand mu, the generation area of black streaked dwarf virus of rice in 2008 then surpasses 4,000,000 mu, and the situation of disease eruption and prevalence is triggered at any moment.
Black streaked dwarf virus of rice is the viroses of plant of propagating with small brown rice planthopper, still pasts medical help the emergent strategies that prevent that adopt " controlling the worm diseases prevention " in the production in case morbidity is caused harm more.And the band poisonous insect of small brown rice planthopper amount be black streaked dwarf virus of rice popular main affecting factors takes place, therefore setting up a kind of rapid sensitive, to detect small brown rice planthopper be very necessary with the method for malicious rate for the popular and disease control of prediction black streaked dwarf virus of rice etc.Be used at present to detect small brown rice planthopper in the world and comprise ELISA, RT-PCR, Northern hybridization etc., but RT-PCR and Northern cost height are unsuitable for the mass detection sample with the method for malicious rate; How anti-the preparation RBSDV RBSDV detection methods of setting up the spot immune method have rapid sensitive, simple characteristics, be expected to detect a kind of RBSDV detection technique that China basic unit agriculture technical staff is used that is suitable for, carry out early detection and early warning in time in early days, can also be applied to the research of the basic field of interaction between RBSDV, small brown rice planthopper and the plant host three simultaneously the band poisonous insect.
Summary of the invention
The purpose of this invention is to provide a kind of detection kit that is exclusively used in the interior rice black-streaked dwarf virus of small brown rice planthopper body in the single head small brown rice planthopper.
Black streaked dwarf virus of rice detection kit of the present invention comprises:
1.RBSDV polyclonal antibody: RBSDV S10 gene prokaryotic protein immunization rabbit acquisition polyclonal antibody (1:3000～1:5000), per unit 1 μ l;
2. ELIAS secondary antibody: the goat-anti rabbit of alkaline phosphatase lipase mark, its work are tired to (1:5000～1:10000), per unit 3-5 μ l is used to detect the RBSDV that small brown rice planthopper is carried;
3. nitrocellulose filter;
4. coating buffer: carbonate buffer solution (0.05M, the pH value is 9.6);
5. washing lotion: phosphate tween damping fluid (PBST) (1000ml 0.01M pH value is to add 0.5ml Tween-20 among 7.5 the PBS);
6. confining liquid: 1% skimmed milk power (0.01M PBST) or 2% bovin serum albumin (BSA) (0.01M PBST);
7. colour developing liquid: 6mg 4-chloro-1-naphthols, 2ml absolute ethyl alcohol, 10ml 0.02M PBS (pH7.4), 7 μ l 30%H
2O
2Face and use preceding mixing, be used to detect the RBSDV that small brown rice planthopper is carried;
The use of kit provided by the invention is divided into standard program and simplifies procedures.
1. rice black-streaked dwarf virus kit standard program in the single head small brown rice planthopper, concrete steps are:
1.1 the preparation of small brown rice planthopper
Select the detection that 2 ages, above single head small brown rice planthopper was used to carry rice black-streaked dwarf virus, small brown rice planthopper is placed centrifuge tube, mash, add 100 μ L coating buffers with toothpick, centrifugal (5000r/min) 3min, it is standby to get supernatant (small brown rice planthopper extract).
1.2 sealing
With pencil nitrocellulose filter (NC) is divided into the grid square of 5mm * 5mm in advance, gets supernatant point that 3 μ L 4.1 obtain on the NC film in the grid square (half is as the criterion not exceed grid square); Respectively get simultaneously the rice black-streaked dwarf virus of purifying about 3 μ L and distillation water spot on film in advance in the good grid square of mark, as positive control and negative control, room temperature is dried film respectively.The film of drying is placed plastic casing or glass culture dish, add 1% skimmed milk power or 2% bovin serum albumin (BSA) confining liquid, require the complete submergence of film is jiggled for 37 ℃ sealing 30min.
1.3 hatch
Push down film with tweezers and abandon confining liquid, with rice black-streaked dwarf virus polyclonal antibody and confining liquid by (the dilution proportion preparation Incubating Solution of 1:5000～1:10000) immerses Incubating Solution fully with film, jiggles for 37 ℃, hatches (1～1.5) h.Push down film with tweezers and abandon Incubating Solution, add phosphate Tween20 damping fluid,, jiggle, wash film (3～5) min, push down film with tweezers and abandon washing lotion, repeat to wash film three times the complete submergence of film.
1.4 two anti-hatching
Push down film with tweezers and abandon washing lotion, with two anti-and confining liquids of horseradish peroxidase-labeled by (the dilution proportion preparation Incubating Solution of 1:2000～1:3000) immerses two anti-Incubating Solutions fully with film, jiggles for 37 ℃, hatch (1.5～2) h, push down film with tweezers again and abandon two anti-Incubating Solutions.Add phosphate Tween20 damping fluid,, jiggle, wash film (3～5) min, push down film with tweezers and abandon washing lotion, repeat to wash film three times the complete submergence of film.
1.5 colour developing
Push down film with tweezers and abandon washing lotion, add colour developing liquid, 37 ℃ leave standstill colour developing 30min.With the distilled water flushing film, room temperature is dried.
1.6 result
At first observe the color reaction of contrast after the colour developing, positive control should show bluish violet, and negative control should not develop the color, and the color reaction of observation sample again if show bluish violet, assert that then this small brown rice planthopper carries rice black-streaked dwarf virus; If do not develop the color, assert that then this small brown rice planthopper do not carry rice black-streaked dwarf virus.
2. the single head small brown rice planthopper is carried the rice black-streaked dwarf virus kit and simplifies procedures, and concrete steps are:
2.1 the preparation of small brown rice planthopper
Select the detection that 2 ages, above single head small brown rice planthopper was used to carry rice black-streaked dwarf virus, small brown rice planthopper is placed centrifuge tube, mash with toothpick, add 100 μ L bag and be cushioned liquid and shake up, supernatant is standby.
2.2 sealing
With pencil nitrocellulose filter (NC) is divided into the grid square of 5mm * 5mm in advance, dips in a little 5.1 supernatant point that obtain on the NC film in the grid square (half is as the criterion not exceed grid square) with toothpick; Dip in the rice black-streaked dwarf virus (the also available this method that adopted is determined as positive band poison small brown rice planthopper extract) of a little purification with toothpick simultaneously and distill water spot on film in advance in the good grid square of mark, as positive control and negative control, room temperature is dried film respectively.The film of drying is placed plastic casing or glass culture dish, add 1% skimmed milk power or 2% bovin serum albumin (BSA) confining liquid, require the complete submergence of film, (15 ℃～30 ℃) jiggle under the room temperature condition, sealing 60min.
2.3 hatch
Push down film with tweezers and abandon confining liquid, with rice black-streaked dwarf virus polyclonal antibody and confining liquid by (the dilution proportion preparation Incubating Solution of 1:5000～1:10000) immerses Incubating Solution fully with film, jiggles under the room temperature condition, hatches 3h.Push down film with tweezers and abandon Incubating Solution, add phosphate Tween20 damping fluid,, jiggle, wash film (3～5) min, push down film with tweezers and abandon washing lotion, repeat to wash film three times the complete submergence of film.
2.4 two anti-hatching
Push down film with tweezers and abandon washing lotion, with two anti-and confining liquids of horseradish peroxidase-labeled by (the dilution proportion preparation Incubating Solution of 1:2000～1:3000) immerses two anti-Incubating Solutions fully with film, jiggles under the room temperature condition, hatch 3h, push down film with tweezers again and abandon two anti-Incubating Solutions.Add phosphate Tween20 damping fluid,, jiggle, wash film (3～5) min, push down film with tweezers and abandon washing lotion, repeat to wash film three times the complete submergence of film.
2.5 colour developing
Push down film with tweezers and abandon washing lotion, add colour developing liquid, leave standstill colour developing (1～2) h under the room temperature condition.With the distilled water flushing film, room temperature is dried.
2.6 result
At first observe the color reaction of contrast after the colour developing, when negative control does not show bluish violet if positive control manifests bluish violet, observe the color reaction of test sample,, assert that then this small brown rice planthopper carries rice black-streaked dwarf virus if show bluish violet; If do not show bluish violet, assert that then this small brown rice planthopper do not carry rice black-streaked dwarf virus.
The principle of this patent is a strong Electrostatic Absorption power of utilizing nitrocellulose filter to have, biomacromolecule such as adsorbed proteins effectively under neutrallty condition.Thereby, antigen is adsorbed in nitrocellulose filter after, just can utilize film to carry out antigen-antibody reaction as solid phase carrier.After adding antibody, can combine with the antigen on the film, and then add the antiantibody that has label, make mark crosslinked indirectly on cellulose membrane by the combination of antiantibody and corresponding antibodies.After adding the corresponding substrate of label at last, label can form insoluble product with substrate-function, present mottled painted, thereby result of determination.Do not appear in the newspapers as yet up to now and use in the detection of this method rice black-streaked dwarf virus in the single head small brown rice planthopper.
Utilize black streaked dwarf virus of rice (RBSDV) specific polyclonal antibody to set up a kind of spot immune (DIBA) detection method, built up the detection kit of black streaked dwarf virus of rice with this method, its main agents all provides with working fluid or concentrate form, have easy to use, simple to operate, detect rapid sensitive, the result waits characteristics accurately and reliably.
The more original RT-PCR of the detection method of the rice black-streaked dwarf virus that provides in the kit of the present invention, Northern blot and conventional ELASA detection method have more economically, easy and characteristics efficiently.The use of black streaked dwarf virus of rice detection kit of the present invention simultaneously is divided into standard program and simplifies procedures, and can widen the usable range and the suitable crowd of this kit greatly, is convenient to this technology popularization is applied to grass-roots unit.Black streaked dwarf virus of rice is simplified procedures and can be carried out detection under the non-laboratory condition that does not need instruments such as hydro-extractor, shaking table in addition, and this there is no report in worldwide.
Description of drawings
Fig. 1 is with malicious rate result schematic diagram (every grid represent a small brown rice planthopper, and it is malicious for band to be bluish violet, otherwise for not being with poison) for this patent provides the kit standard program to detect the single head small brown rice planthopper
Specific implementation method
Embodiment 1, and standard program detects small brown rice planthopper and carries rice black-streaked dwarf virus
In June, 2008, long-term black streaked dwarf virus of rice generating region was gathered inferior small brown rice planthopper more than 3 ages in age from Xinghua, Jiangsu Province county, adopt the immune reagent kit standard detecting method of rice black-streaked dwarf virus in the single head small brown rice planthopper, obtain result such as Fig. 1, show that it is feasible that this method detects, it can specific detection go out whether contain rice black-streaked dwarf virus in the single head small brown rice planthopper.
The present invention mainly serves the quick and precisely detection of black streaked dwarf virus of rice paddy rice and band kissing bug, can make basic unit and quarantine departments in time disease be carried out the prediction early warning and be formulated the general control strategy, the further diffusion that can prevent and treat the small brown rice planthopper of high-band poison rate is got food and is caused disaster, and can reduce increasing the weight of of this increase of farming that blindly medication causes and environmental pollution again.This technology also has very big using value at aspects such as the resistance evaluation of virus and the interaction that passes virus mediator, kind and genetics of resistance analyses simultaneously.In time fill up in view of one that does not also have both at home and abroad a kind of detection kit that agrotechnical department of basic unit used and had the black streaked dwarf virus of rice of independent intellectual property right that is suitable for, the present invention to can be described as current domestic and international present situation.
Above-mentioned enforcement does not limit the present invention in any form.
Claims (2)
1, a kind of immunity detection reagent that is used for single head small brown rice planthopper rice black-streaked dwarf virus is characterized in that comprising rice black-streaked dwarf virus polyclonal antibody, ELIAS secondary antibody, nitrocellulose filter, coating buffer, confining liquid, washing lotion and colour developing liquid.
2, the standard program of the immunity detection reagent of rice black-streaked dwarf virus and simplifying procedures in the single head small brown rice planthopper; It is characterized in that: 1) described standard program is to have carried out having under the instrument conditions such as hydro-extractor and shaking bath; 2) described standard program is not need instruments such as hydro-extractor and shaking table to operate to finish.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101713001B (en) * | 2009-12-09 | 2011-01-26 | 湖南农业大学 | RT-PCR detection method of south rice black-streaked dwarf virus (SRBSDV) |
| CN101713000B (en) * | 2009-12-09 | 2011-04-06 | 湖南农业大学 | RT-PCR detection method of ordinary rice black streaked dwarf virus (RBSDV) |
| CN102168151A (en) * | 2011-03-24 | 2011-08-31 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting rice black-streaked dwarf viruses in plant hopper |
| CN102168150A (en) * | 2011-03-24 | 2011-08-31 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting southern rice black-streaked dwarf viruses in plant hopper |
| CN103103292A (en) * | 2013-01-31 | 2013-05-15 | 江苏省农业科学院 | Method for rapidly detecting in-vivo rice black streaked dwarf virus of single-head small brown rice planthopper |
| CN103760346A (en) * | 2014-01-25 | 2014-04-30 | 云南省农业科学院生物技术与种质资源研究所 | Dot fluorescence immunoassay method for quantitatively detecting plant virus |
| 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 |
| CN104345151A (en) * | 2013-08-08 | 2015-02-11 | 北京和杰创新生物医学科技有限公司 | Membrane blocking method improving specificity of enzyme-linked immunospot assay |
| CN105675875A (en) * | 2016-02-22 | 2016-06-15 | 浙江大学 | Colloidal gold immunity test strip for fast detecting southern rice black-streaked dwarf viruses and preparing method thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101713001B (en) * | 2009-12-09 | 2011-01-26 | 湖南农业大学 | RT-PCR detection method of south rice black-streaked dwarf virus (SRBSDV) |
| CN101713000B (en) * | 2009-12-09 | 2011-04-06 | 湖南农业大学 | RT-PCR detection method of ordinary rice black streaked dwarf virus (RBSDV) |
| CN102168151A (en) * | 2011-03-24 | 2011-08-31 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting rice black-streaked dwarf viruses in plant hopper |
| CN102168150A (en) * | 2011-03-24 | 2011-08-31 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting southern rice black-streaked dwarf viruses in plant hopper |
| CN102168151B (en) * | 2011-03-24 | 2013-01-09 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting rice black-streaked dwarf viruses in plant hopper |
| CN102168150B (en) * | 2011-03-24 | 2013-04-10 | 江苏省农业科学院 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapidly detecting southern rice black-streaked dwarf viruses in plant hopper |
| CN103103292A (en) * | 2013-01-31 | 2013-05-15 | 江苏省农业科学院 | Method for rapidly detecting in-vivo rice black streaked dwarf virus of single-head small brown rice planthopper |
| CN104345151A (en) * | 2013-08-08 | 2015-02-11 | 北京和杰创新生物医学科技有限公司 | Membrane blocking method improving specificity of enzyme-linked immunospot assay |
| CN104345151B (en) * | 2013-08-08 | 2016-01-06 | 北京和杰创新生物医学科技有限公司 | Improve the specific membrane enclosure method of elispot assay |
| CN103760346A (en) * | 2014-01-25 | 2014-04-30 | 云南省农业科学院生物技术与种质资源研究所 | Dot fluorescence immunoassay method for quantitatively detecting plant virus |
| CN103760346B (en) * | 2014-01-25 | 2015-06-24 | 云南省农业科学院生物技术与种质资源研究所 | Dot fluorescence immunoassay method for quantitatively detecting plant virus |
| 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 |
| CN105675875A (en) * | 2016-02-22 | 2016-06-15 | 浙江大学 | Colloidal gold immunity test strip for fast detecting southern rice black-streaked dwarf viruses and preparing method thereof |
| CN105675875B (en) * | 2016-02-22 | 2017-06-30 | 浙江大学 | A kind of colloid gold immune test paper bar of quick detection southern rice black-streaked dwarf virus and preparation method thereof |
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Application publication date: 20090513 |