CN1828304A - Rice stripe virus immune detection reagent kit - Google Patents
Rice stripe virus immune detection reagent kit Download PDFInfo
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- CN1828304A CN1828304A CN 200610038187 CN200610038187A CN1828304A CN 1828304 A CN1828304 A CN 1828304A CN 200610038187 CN200610038187 CN 200610038187 CN 200610038187 A CN200610038187 A CN 200610038187A CN 1828304 A CN1828304 A CN 1828304A
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Abstract
Wherein, building a DIPA detection method with the RSV specific monoclonal antibody, and hereby building the reagent box with main reagent provided as working liquid or condensed liquid. This invention can detect whether there is RSV rapidly, and has wide application.
Description
The present invention relates to a kind of immunity detection reagent of rice stripe virus and the application on agricultural thereof, belong to the agricultural cience and farming techniques field.
Background technology
The stripe disease that rice stripe virus (RSV) causes found that all there is generation the back in Korea, Ukraine and China in the Japanese Northeast early than 1897.China from 1962 since this virus is found in the Jiangsu and Zhejiang Provinces, the whole nation 16 provinces popular.This disease at present in Yunnan, Liaoning, Beijing, Henan, Shandong, Jiangsu, Shanghai are still very common, the particularly Golconda in Yunnan, Chuxiong, Kunming, Pekinese's doube bridge, the Yuanyang, Henan, ground such as the Jiangyan City in Jining, Shandong and the north, Jiangsu, Hongze take place more general.
Its main cause that continues to break out is: warm winter weather in recent years helps surviving the winter of infection insect small brown rice planthopper, and small brown rice planthopper poison source radix is accumulated for many years, adds that easy susceptible rice varieties plants in a large number.According to incompletely statistics, only Jiangsu Province's stripe disease generation area in 2004 just reaches 2,700 ten thousand mu, accounts for 79% of Jiangsu Province's rice area, and the expert estimates that stripe disease will be in after 2006 5 years and lay particular stress on occurrence tendency that prevention work is very urgent.
And stripe disease is the viroses of plant of propagating with small brown rice planthopper, in case morbidity is caused harm and is still pasted medical help, so the control of this disease should serve as crucial i.e. " controlling the worm diseases prevention " with the control small brown rice planthopper. and the band poisonous insect of small brown rice planthopper amount be stripe disease 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 stripe disease 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; Preparation RSV monoclonal antibodies such as Zhou Yijun are used for virus and detect, the RSV detection method of setting up the spot immune method based on this has rapid sensitive, simple characteristics, be expected to detect a kind of RSV 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 RSV, small brown rice planthopper and the plant host three simultaneously the band poisonous insect.
Summary of the invention
The detection kit that the purpose of this invention is to provide rice stripe virus in a kind of small brown rice planthopper body.
Rice stripe virus detection kit of the present invention comprises:
1) RSV monoclonal antibody: RSV immune mouse acquisition monoclonal antibody (1: 5000-20000), per unit 100ul;
2) ELIAS secondary antibody A: it is 1 that the sheep anti-mouse igg of horseradish peroxidase (HRP) mark, its work are tired: 5000-10000, and per unit 100ul is used to detect the RSV that small brown rice planthopper is carried;
3) ELIAS secondary antibody B: it is 1 that the sheep anti-mouse igg of alkaline phosphatase (AP) mark, its work are tired: 100000-300000, and per unit 100ul is used to detect the RSV that plant tissue carries;
4) nitrocellulose filter;
5) sample preparation liquid: phosphate buffer (PBS) (0.01M, pH value 7.5);
6) washing lotion: phosphate tween damping fluid (PBST) (adding 0.5mlTween-20 among the 1000ml 0.01M PBS);
7) confining liquid: 1% skimmed milk power (0.01M PBST) or 2% bovin serum albumin (BSA) (0.01M PBST);
8) colour developing liquid A:12mg 4-chloro-1-naphthols, 4ml 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 RSV that small brown rice planthopper is carried;
9) colour developing liquid B:BCIP/NBT is used to detect the RSV that plant tissue carries;
RSV detection kit at room temperature (4-25 ℃) using method is:
1) with pure water washing lotion is diluted 10 times to 0.01M, the configuration confining liquid is standby.
2) nitrocellulose filter (NC) film pencil drawing 0.25cm
2About grid square.
3) single head small brown rice planthopper or plant tissue to be measured 1 gram are put and are added 2 sample preparation liquid in the 0.2ml centrifuge tube, smash to pieces with toothpick, get the about 3 μ L of supernatant 1 droplet or repeat 3-4 point sample in nitrocellulose filter grid square central authorities with toothpick, and room temperature is dried.
4) film with drying immerses in the confining liquid room temperature 1h.
5) at the monoclonal antibody of a unit of 10ml confining liquid adding, film is taken out immersion, room temperature is placed 3h.
6) take out film, washing lotion is washed 3 times, each 3-5min.
7) get two anti-A of a unit or B and add in the 10ml confining liquid, film is taken out immerse, room temperature is placed 3h.
8) take out the film washing lotion and wash 3 times, each 3min.
9) film is immersed among the colour developing liquid A or B of new preparation, room temperature is to colour developing (1-2h).
10) after the colour developing, with the tap water flushing, room temperature is dried.
11) determine the band poison situation of sample according to the color in the grid, be blue purple for the band poison, colourless for not being with poison.
RSV detection kit (37 ℃) using method under laboratory condition is:
1) with pure water washing lotion is diluted 10 times to 0.01M, the configuration confining liquid is standby.
2) nitrocellulose filter (NC) film pencil drawing 0.25cm
2About grid square.
3) single head small brown rice planthopper or plant tissue to be measured 1 gram are put and are added 2 sample preparation liquid in the 0.2ml centrifuge tube, smash to pieces with toothpick, and the centrifugal 3min of 5000rpm gets supernatant 3 μ L points in NC film grid square, the room temperature airing.
4) film with drying immerses in the confining liquid 37 ℃ of shaking tables, 30min.
5) at the monoclonal antibody of a unit of 10ml confining liquid adding, film is taken out immersion, 37 ℃ of shaking tables, 1-1.5h.
6) take out film, washing lotion is washed 3 times, each 3min.
7) get two anti-A of a unit or B and add in the 10ml confining liquid, film is taken out immerse 37 ℃ of shaking tables, 1.5h-2h.
8) take out the film washing lotion and wash 3 times, each 3min.
9) film is immersed among the colour developing liquid A or B of new preparation 37 ℃ of shaking tables, 30min.
10) after the colour developing, with tap water flushing, room temperature airing.
11) determine the band poison situation of sample according to the color in the grid, be blue purple for the band poison, colourless for not being with poison.
Description of drawings
Fig. 1 is with malicious rate result schematic diagram for the DIBA method detects small brown rice planthopper
(A is the disease leaf to Fig. 2, and B is the nontoxic small brown rice planthopper of single head, and a～e is respectively different single head bands poison small brown rice planthoppers for DIBA detects disease leaf and single head band poison small brown rice planthopper result schematic diagram; 1-11 is that different extension rates are handled, and respectively is 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times, 6400 times, 12800 times, 25600 times, 51200 times, 102400 times and 204800 times)
Fig. 3 is 37 ℃ of following monoclonal antibody incubation time test findings synoptic diagram (1-6 represent respectively sample hatch 0.5h in monoclonal antibody, 1h, 1.5h, 2h, 2.5h, six kinds of processing of 3h)
Fig. 4 is 37 ℃ of down two anti-incubation time test findings synoptic diagram (I-VI represent respectively sample hatch 0.5h, 1h, 1.5h, 2h, 2.5h, six kinds of processing of 3h in anti-two)
Specific implementation method
Embodiment 1,
Be with malicious situation to detect to the vector small brown rice planthopper, the step under the room temperature is:
1) with pure water washing lotion is diluted 10 times to 0.01M, the configuration confining liquid is standby.
2) nitrocellulose filter (NC) film pencil drawing 0.25cm
2About grid square.
3) the single head small brown rice planthopper is put and adds 2 sample preparation liquid in the 0.2ml centrifuge tube, smashs to pieces with toothpick, gets the about 3 μ L point samples of supernatant 1 droplet in nitrocellulose filter grid central authorities, and room temperature is dried.
4) film with drying immerses in the confining liquid room temperature 1h.
5) at the monoclonal antibody of a unit of 10ml confining liquid adding, film is taken out immersion, room temperature is placed 3h.
6) take out film, washing lotion is washed 3 times, each 3-5min.
7) get the two anti-A of a unit and add in the 10ml confining liquid, film is taken out immerse, room temperature is placed 3h.
8) take out the film washing lotion and wash 3 times, each 3min.
9) film is immersed among the colour developing liquid A of new preparation, room temperature is to colour developing (1-2h).
10) after the colour developing, with the tap water flushing, room temperature is dried.
11) test findings such as Fig. 1 determine the band poison situation of sample to be blue purple for the band poison according to the color in the grid, and be colourless for not being with poison.
The test of diluted sample degree
Single head small brown rice planthopper and sick leaf become 11 gradients since 100 times by doubling dilution, do the negative contrast of same dilutability with nontoxic small brown rice planthopper.
The result shows, the single head small brown rice planthopper is diluted to 25600 times of still visible faint positive reactions, but it is the strongest to dilute 100 times of positive reactions, and conventional sense adopts dilution 100-200 doubly more; Sick leaf is diluted to 51200 times of still visible faint positive reactions, but it is better to dilute 400 times of range estimation effects, and conventional sense adopts dilution 200-500 doubly (Fig. 2) more.But preferably adopt fresh incidence of leaf or employing to detect the method for plant tissue.
The test of antibody incubation time
Monoclonal antibody working concentration 1: 20000 is hatched 0.5h, 1h, 1.5h, 2h, 2.5h, 3h (two anti-working concentrations 1: 2500, the two anti-1.5h of hatching) under 37 ℃ respectively, to determine to change the influence of monoclonal antibody incubation time to testing result.The result shows 37 ℃ when hatching 0.5h, just can detect positive, and the spot colors when hatching 1h is as broad as long substantially when hatching 1.5h, 2h, 2.5h, color slightly descend (Fig. 3) when hatching 3h.So determine that it is best incubation time that monoclonal antibody is hatched 1～1.5h for 37 ℃.
Determine monoclonal antibody working concentration 1: 20000, and hatch 1.5h under 37 ℃; Two anti-working concentrations 1: 2500 are hatched 0.5h, 1h, 1.5h, 2h, 2.5h, 3h respectively under 37 ℃, to determine to change the influence of two anti-incubation times to testing result.The result shows 37 ℃ when hatching 0.5h, basic detection does not go out positive, spot colors when hatching 1h is more shallow, hatch 1.5h and can obviously detect positive, hatch 2h, 2.5h the dark and colour developing degree basic identical (Fig. 4) of color during 3h is so determine that two resist 37 ℃ to hatch 1.5h-2h be best incubation time.
The test of kit storage life
The kit preservation condition is 4 ℃, through 6 months mensuration, uses kit and can obtain tangible testing result.Consider monoclonal antibody and two anti-thermal adaptabilities in the kit, require kit when transportation, must adopt the low temperature means of transportation.
The present invention mainly serves the quick and precisely detection of rice stripe virus 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.Simultaneously this technology in the resistance evaluation and the genetics of resistance analysis of virus and the interaction that passes virus mediator, kind, be suspected to be that the aspects such as diagnosis of case also have very big using value.Also do not have a kind of detection kit that agrotechnical department of basic unit used and had the rice stripe virus of independent intellectual property right that is suitable in view of domestic, of can be described as current present status in China of the present invention in time fills up.
Above-mentioned enforcement does not limit the present invention in any form.
Claims (8)
1, a kind of immunity detection reagent that is used to detect rice stripe virus is characterized in that comprising rice stripe virus monoclonal antibody specific and ELIAS secondary antibody.
2, the immunity detection reagent of rice stripe virus according to claim 1 is characterized in that this kit also comprises nitrocellulose filter, sample preparation liquid, confining liquid, washing lotion and colour developing liquid.
3, the immunity detection reagent of rice stripe virus according to claim 1 and 2 is characterized in that the monoclonal antibody in the mouse source of described rice stripe virus.
4, the immunity detection reagent of rice stripe virus according to claim 1 and 2 is characterized in that described two anti-sheep anti-mouse igg serum preparations for horseradish peroxidase (HRP) or alkaline phosphatase (AP) mark.
5, the immunity detection reagent of rice stripe virus according to claim 1 and 2 is characterized in that described washing lotion is to contain 0.5% phosphate buffer.
6, the immunity detection reagent of rice stripe virus according to claim 1 and 2 is characterized in that described colour developing liquid is 4-chloro-1-naphthols, absolute ethyl alcohol, PBS (pH7.4) and 30%H
2O
2Mixed liquor or BCIP/NBT.
7, the immunity detection reagent of rice stripe virus according to claim 1 and 2 is characterized in that described kit all can use under laboratory and non-laboratory condition.
8, the immunity detection reagent of rice stripe virus according to claim 1 and 2 is characterized in that described kit can be 4 ℃ of following conditions preservations 6 months.
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| CN 200610038187 CN1828304A (en) | 2006-02-09 | 2006-02-09 | Rice stripe virus immune detection reagent kit |
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| CN 200610038187 CN1828304A (en) | 2006-02-09 | 2006-02-09 | Rice stripe virus immune detection reagent kit |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182586B (en) * | 2007-12-03 | 2010-06-02 | 江苏省农业科学院 | RAPD marker tightly linking gene resistance to rice sheath blight |
| 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 |
| CN104267186A (en) * | 2014-10-16 | 2015-01-07 | 云南农业大学 | Method for detecting rice stripe virus in single-head laodelphax striatellus through dot-ELISA (dot-enzyme-linked immuno sorbent assay) |
| CN105388289A (en) * | 2015-11-16 | 2016-03-09 | 江苏省农业科学院 | Method for detecting distribution of rice stripe viruses in plants with immunohistochemical method |
| 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 |
| CN109633152A (en) * | 2019-01-28 | 2019-04-16 | 浙江大学 | A kind of colloid gold immune test paper item and preparation method thereof of quick detection rice stripe virus |
-
2006
- 2006-02-09 CN CN 200610038187 patent/CN1828304A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182586B (en) * | 2007-12-03 | 2010-06-02 | 江苏省农业科学院 | RAPD marker tightly linking gene resistance to rice sheath blight |
| 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 |
| CN104267186A (en) * | 2014-10-16 | 2015-01-07 | 云南农业大学 | Method for detecting rice stripe virus in single-head laodelphax striatellus through dot-ELISA (dot-enzyme-linked immuno sorbent assay) |
| CN104267186B (en) * | 2014-10-16 | 2016-06-08 | 云南农业大学 | Dot-ELISA is utilized to detect the method for rice stripe virus in single head small brown rice planthopper body |
| CN105388289A (en) * | 2015-11-16 | 2016-03-09 | 江苏省农业科学院 | Method for detecting distribution of rice stripe viruses in plants with immunohistochemical method |
| 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 |
| CN109633152A (en) * | 2019-01-28 | 2019-04-16 | 浙江大学 | A kind of colloid gold immune test paper item and preparation method thereof of quick detection rice stripe virus |
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