CN104111331A - Antibody chip for detecting compositae plant virus diseases, and kit and method of antibody chip - Google Patents
Antibody chip for detecting compositae plant virus diseases, and kit and method of antibody chip Download PDFInfo
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Abstract
The invention relates to an antibody chip for screening and diagnosing compositae plant virus diseases, and a kit and a method of the antibody chip. The antibody chip for screening and diagnosing compositae plant virus diseases is mainly characterized by comprising a base membrane and specific capture antibodies (primary antibodies) which are respectively pointed on the base membrane and used for capturing seven types of viruses with harm to the compositae plant, wherein the antibodies (primary antibodies) for capturing seven types of viruses have resistance to bean yellow mosaic virus, resistance to arabis mosaic virus, resistance to curtobacterium flaccumfaciens virus, resistance to cucumber mosaic virus, resistance to tobacco rattle virus, resistance to tobacco ringspot virus and resistance to tomato spotted wilt virus; three positive quality control points P, IgG pointed to be alkaline phosphatase mark, three negative quality control points N and a carbonate buffer solution pointed to reach a pH being 9.2-9.8 are also encapsulated on the base membrane. The antibody chip for screening and diagnosing compositae plant virus diseases has the advantages that the antibody chip for detecting a plurality of viruses of the compositae plant is successfully built, so that comprehensive detection is carried out on a plurality of pathogens at a high flux.
Description
Technical field
The present invention relates to a kind of examination of the viroses of plant and the antibody chip of diagnosis and kit and method thereof of being applied to, bean virus 2, arabis mosaic virus, bean bacterial northern wilt poison, cucumber mosaic virus, Tobacco rattle virus, nepovirus and 7 kinds of viruses of tomato spotted wilf virus mainly for feverfew, belong to biological technical field.
Background technology
In feverfew, have medicinal in a large number, view and admire and economic plants, virosis is one of key constraints of feverfew production, bean virus 2 (Bean yellow mosaic virus, BYMV), arabis mosaic virus (Arabis mosaic virus, ArMV), bean bacterial northern wilt poison (Broad bean wilt virus, BBWV), cucumber mosaic virus (Cucumber mosaic virus, CMV), Tobacco rattle virus (Tobacco rattle virus, TRV), nepovirus (Tobacco ringspot virus, TRSV) and tomato spotted wilf virus (Tomato spotted wilt virus, TSWV) all can infect feverfew.BYMV infects the various plants such as composite family, Chenopodiaceae, Amaranthaceae, Aizoaceae; This virus communication media is mainly aphid, in perishability mode, propagates, and also can propagate by mechanical inoculation.ArMV is the quarantine harmful organisms of the inward plant of China, can endanger approximately 174 and belong to 200 various plants, comprises composite vegetable, flowers etc., causes the symptoms such as floral leaf, yellow chlorisis, dwarfing, shrinkage, necrosis; This virus is propagated by approach such as nematode and seeds.BBWV host range is wide, can infect 328 Plants in 186 genus of 44Ge section, comprises multiple feverfew; Cause the symptoms such as floral leaf, dwarfing, wilting and plant be withered; Occurring in nature is propagated in perishability mode by multiple aphid, and The book of Changes mechanical inoculation is propagated.CMV is worldwide distribution virus, can infect more than 1000 kind of unifacial leaf and dicotyledon, be host plant at most, distribute the most extensively, the plant virus of tool Economic Importance; CMV mainly relies on multiple aphid for passing malicious medium, in perishability mode, propagates, and easily by mechanical inoculation, propagates, and the seed of Chinese dodder also can pass poison.TRV host range is wide, can infect 400 kinds of dicotyledonous and monocotyledons of 50 section, under natural conditions, can infect the vegetables and flowers such as sunflower, lettuce; Occurring in nature is mainly propagated by intending burr nematode and burr nematode, and water, seed or the seed of Chinese dodder also can pass poison.TRSV is the quarantine harmful organisms that can propagate with vegetable seeds, nursery stock, 54 section's 300 various plants that can cause harm, and natural infection host comprises multiple composite vegetable, flowers etc.; Be subject to Symptoms that TRSV infects different because of host, there are ring-type or wire line, chlorisis spot, necrotic plaque etc. in blade conventionally; This virus is propagated by approach such as nematode, mechanical inoculation and seeds, and it is many that kind passes host, and seed dispersal is the main path of TRSV long-distance communications.TSWV is the inward plant quarantine harmful organism of China, and host range is extensive, at least infects 900 Plants, wherein multiple feverfew easy infection TSWV; This virus mainly passes poison by juice, and seed can pass poison, easily by thrips, in persistence mode, propagates, and grafting also can pass poison.
Feverfew is subject to virosis to infect the multiple symptom of rear performance, as floral leaf, deformity, yellow, stunt, downright bad etc., diseased plant depauperation, causes yield and quality greatly to reduce.In field, feverfew infects after virosis, it is very large that its symptom is affected by kind, plant growing stage, environmental baseline and virus strain, therefore be difficult to judge from Symptoms virosis by any or which kind virus is caused actually, therefore the synchronization detecting method of high flux, highly sensitive multiple feverfew virus, for finding out in time cause of disease, eliminate primary source of infection, preventing that its spread in china of virosis is most important.Application at present more widely plant virus detection method has enzyme linked immunosorbent assay (ELISA) experiment (Enzyme Linked Immuno Sorbent Assay, ELISA) and reverse transcription polymerase chain amplification (Reverse Transcription-PCR, RT-PCR), but adopt conventional method to detect a plurality of cause of diseases in sample, process is very long, need to spend a large amount of human and material resources and financial resources.
Antibody chip be utilize between antigen and antibody molecule can specific binding principle, capture antibody is arranged with array pattern point sample, after analyzed example reaction, obtain hybridization signal, finally determine distribution and the content information of target molecule in sample.Antibody chip has can carry out high flux parallel analysis, saving reagent and material, the simple feature of sample preparation process, has been applied to a plurality of life sciences such as protein function research, medical diagnosis.Adopt antibody chip technology for detection feverfew virosis, once experiment can detect multiple virus simultaneously, can improve detection efficiency, shortens sense cycle, reduces testing cost.
Summary of the invention
The object of the invention is to avoid the deficiencies in the prior art part and the antibody chip that is applied to detect feverfew virosis is provided, that the described this antibody chip of diagnosing 7 kinds of feverfew viruses for combining has is easy, quick, sensitive, specificity is good, the feature of flux.
An also object of the present invention is to provide a kind of antibody chip kit that is applied to detect feverfew virosis.
Another object of the present invention is a kind of antibody chip detection method that is applied to detect feverfew virosis.
For achieving the above object, the technical scheme that the present invention takes is: a kind of antibody chip that is applied to detect feverfew virosis, it is characterized in that, 7 kinds of viral specificity capture antibodies (primary antibodie) of the harm feverfew that includes basement membrane and make respectively on basement membrane, described 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; On basement membrane, be also packaged with 3 positive quality control point P, point is made as the IgG of alkali phosphatase enzyme mark and is packaged with 3 negative Quality Control point N, and point is made as the carbonate buffer solution of pH9.2-9.8.The described preparation method who is applied to detect the antibody chip of feverfew virosis, the steps include:
(1) antibody dilution: with capture antibody (primary antibodie) dilution buffer liquid dilution 7 kinds of polyclone capture antibodies (primary antibodie), capture antibody after dilution (primary antibodie) concentration is 1/18-1/3 times of antibody stoste, it is BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV are followed successively by antibody stoste for the antibody concentration of point sample 1:17,1:14,1:17,1:14,1:15,1:3 and 1:15 times, on chip, add the anti-goat IgG stoste of alkali phosphatase enzyme mark donkey of 1:10 dilution as the positive quality control point of chip, chip sampling liquid is as negative Quality Control point simultaneously; Point sample damping fluid is: sodium carbonate Na
2cO
31.59g, sodium bicarbonate NaHCO
32.93g, sodium azide NaN
30.2g, distilled water ddH
2o 1000mL, pH 9.6;
(2) point sample: by after cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 ℃ of constant temperature ovens.Press chip dot matrix, every kind of polyclone capture antibody (primary antibodie) repeats 2 points of system; Respectively for 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus put the position at numbering 1-7 successively; Positive quality control, negative Quality Control are put respectively in position at P and N;
(3) mentioned reagent is pressed to array arrangement point sample in membrane surface, every some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in to wet box, humidity is 45%, 37 ℃ of fixedly 30min;
(4) sealing: the antibody chip fixing is sealed to 1h without 37 ℃ of protein blocking liquid with 1% in wet box, remove after confining liquid, 2-8 ℃ of preservation, is antibody chip finished product.
An antibody chip kit that is applied to detect feverfew virosis, its principal feature is, includes antibody chip; The detection antibody of the alkali phosphatase enzyme mark that 7 kinds of virus is corresponding (two is anti-): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; The positive control of feverfew virus, include the positive control of bean virus 2, the positive control of arabis mosaic virus, the positive control of bean bacterial northern wilt poison, the positive control of the positive control of cucumber mosaic virus, Tobacco rattle virus, the positive control of the positive control of nepovirus, tomato spotted wilf virus; Alkaline phosphatase; Detect antibody (two is anti-) dilution buffer liquid; Lavation buffer solution PBST; The chloro-3-indyl of the bromo-4-of alkaline phosphatase chromogenic substrate 5-phosphate/NBT.
A detection method that is applied to detect the antibody chip kit of feverfew virosis, its principal feature is that step is:
(1) by detection sample and 4 ℃ of overnight hatching of antibody chip; Described antibody chip be include basement membrane and on basement membrane 7 kinds of viral specificity capture antibodies (primary antibodie) of harm feverfew of system respectively, described 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; On basement membrane, be also packaged with 3 positive quality control point P, point is made as the IgG of alkali phosphatase enzyme mark and is packaged with 3 negative Quality Control points, and point is made as the carbonate buffer solution of pH=9.2-9.8;
(2) after washing, add again corresponding plant virus to detect antibody (two is anti-) hybridization and hatch, continue reaction 4h;
(3) after washing, add the colour developing of colour developing working fluid;
Described feverfew virus capture antibody (primary antibodie) is respectively bean virus 2 Bean yellow mosaic virus, BYMV, arabis mosaic virus Arabis mosaic virus, ArMV, bean bacterial northern wilt poison Broad bean wilt virus, BBWV, cucumber mosaic virus Cucumber mosaic virus, CMV, Tobacco rattle virus Tobacco rattle virus, TRV, nepovirus Tobacco ringspot virus, TRSV and tomato spotted wilf virus Tomato spotted wilt virus, at least one in TSWV capture antibody (primary antibodie),
The IgG of usining contrasts as Quality Control.
Principle of the present invention is: choose respectively the specificity capture antibody (primary antibodie) for these 7 kinds of cause of diseases, with array pattern, be fixed on cellulose nitrate basement membrane, utilize cause of disease in capture antibody (primary antibodie) specific binding sample, react with the detection antibody (two is anti-) of alkali phosphatase enzyme mark again, and then in array surface specificity, form the double-antibody sandwich structure of " capture antibody-antigen-detection antibody ", utilize subsequently the specific chromogenic substrate that alkaline phosphatase is corresponding to show hybridization signal, set up a kind of can once detect simultaneously 7 kinds viral efficient, sensitive, easy feverfew method for detecting virus.
The present invention purchases the viral positive control of corresponding detection and polyclone capture antibody (primary antibodie), on 7 identical basement membranes, add mixing positive control, and mix and detect antibody (two is anti-), have no obvious non-specific cross-reaction, establish double antibody sandwich method to each viral sensing range, lowest detectable limit, precision, successfully built the multiple antibody chip detection system that can be used for sample examination.The detectability to actual sample with seed and plant leaf blade extract hot-wire array, result shows that its positive rate is better than or is suitable with ELISA, demonstrates good practical prospect.
Beneficial effect of the present invention: the present invention has successfully built the antibody chip that detects the multiple virus of feverfew, can to a plurality of cause of diseases, carry out comprehensive detection high flux, can be used for the detection monitoring of feverfew virosis, also can be used for the research of feverfew Anti-virus Disease Breeding, there is important Research Significance and social effect.Relatively existing feverfew method for detecting virus, the advantage that antibody chip of the present invention and method have is: (1) device simple, operation is convenient, and cost is low; (2) can realize the detection to a plurality of samples to be checked simultaneously, can improve the diagnosis efficiency of virosis once detect as many as 7 Plants viruses in experiment simultaneously, save human and material resources and financial resources; (3) detection sample preparation is simple, and sample crude extract can meet testing requirement, and testing process does not need specific apparatus and skilled technical ability, is applicable to field and Site Detection; (4) have sensitivity, the specificity equal with methods such as ELISA, promotion prospect is wide.
Accompanying drawing explanation
Fig. 1 is the lattice structure schematic diagram of embodiment 1 antibody chip.
In figure: N represents negative Quality Control; P represents positive quality control.
Fig. 2 is that the testing result of embodiment 2 antibody chip specificity confirmatory experiments shows image.
In figure: 1:BYMV; 2:ArMV; 3:BBWV; 4:CMV; 5:TRV; 6:TRSV; 7:TSWV; 8: water.
Fig. 3 is that embodiment 4 antibody chips detect 7 kinds of viral the results simultaneously.
Embodiment
Below in conjunction with embodiment, principle of the present invention and feature are described, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1: see Fig. 1, a kind of antibody chip that is applied to detect feverfew virosis, it is characterized in that, 7 kinds of viral specificity capture antibodies (primary antibodie) of the harm feverfew that includes basement membrane and make respectively on basement membrane, described 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; On basement membrane, be also packaged with 3 positive quality control point P, point is made as the IgG of alkali phosphatase enzyme mark and is packaged with 3 negative Quality Control point N, and point is made as the carbonate buffer solution of pH 9.2-9.8.
Embodiment 2: described being applied to detects the preparation method of the antibody chip of feverfew virosis, the steps include:
(1) antibody dilution: with capture antibody (primary antibodie) dilution buffer liquid dilution 7 kinds of polyclone capture antibodies (primary antibodie), capture antibody after dilution (primary antibodie) concentration is 1/18-1/3 times of antibody stoste, it is BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV are followed successively by antibody stoste for the antibody concentration of point sample 1:17,1:14,1:17,1:14,1:15,1:3 and 1:15 times, on chip, add the anti-goat IgG stoste of alkali phosphatase enzyme mark donkey of 1:10 dilution as the positive quality control point of chip, chip sampling liquid is as negative Quality Control point simultaneously; Point sample damping fluid is: sodium carbonate Na
2cO
31.59g, sodium bicarbonate NaHCO
32.93g, sodium azide NaN
30.2g, distilled water ddH
2o 1000mL, pH 9.6;
(2) point sample: by after cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 ℃ of constant temperature ovens.Press chip dot matrix, every kind of polyclone capture antibody (primary antibodie) repeats 2 points of system; Respectively for 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus put the position at numbering 1-7 successively; Positive quality control, negative Quality Control are put respectively in position at P and N;
(3) mentioned reagent is pressed to array arrangement point sample in membrane surface, every some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in to wet box, humidity is 45%, 37 ℃ of fixedly 30min;
(4) sealing: the antibody chip fixing is sealed to 1h without 37 ℃ of protein blocking liquid with 1% in wet box, remove confining liquid, 2-8 ℃ of preservation, is antibody chip finished product.
Embodiment 3: a kind of antibody chip kit that is applied to detect feverfew virosis, and its principal feature is, includes antibody chip; The detection antibody of the alkali phosphatase enzyme mark that 7 kinds of virus is corresponding (two is anti-): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; The positive control of feverfew virus, include the positive control of bean virus 2, the positive control of arabis mosaic virus, the positive control of bean bacterial northern wilt poison, the positive control of the positive control of cucumber mosaic virus, Tobacco rattle virus, the positive control of the positive control of nepovirus, tomato spotted wilf virus; Alkaline phosphatase; Detect antibody (two is anti-) dilution buffer liquid; Lavation buffer solution PBST; The chloro-3-indyl of the bromo-4-of alkaline phosphatase chromogenic substrate 5-phosphate/NBT.
Embodiment 4: a kind of detection method that is applied to detect the antibody chip kit of feverfew virosis, and its principal feature is that step is:
(1) by detection sample and 4 ℃ of overnight hatching of antibody chip; Described antibody chip is 7 kinds of viral specificity capture antibodies (primary antibodie) that endanger feverfews that include basement membrane and make respectively on basement membrane, described 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; On basement membrane, be also packaged with 3 positive quality control point P, point is made as the IgG of alkali phosphatase enzyme mark and is packaged with 3 negative Quality Control points, and point is made as the carbonate buffer solution of pH9.2-9.8;
(2) after washing, add again corresponding plant virus to detect antibody (two is anti-) hybridization and hatch, continue reaction 4h;
(3) after washing, add the colour developing of colour developing working fluid:
Described feverfew virus capture antibody (primary antibodie) is respectively bean virus 2 Bean yellow mosaic virus, BYMV, arabis mosaic virus Arabis mosaic virus, ArMV, bean bacterial northern wilt poison Broad bean wilt virus, BBWV, cucumber mosaic virus Cucumber mosaic virus, CMV, Tobacco rattle virus Tobacco rattle virus, TRV, nepovirus Tobacco ringspot virus, TRSV and tomato spotted wilf virus Tomato spotted wilt virus, at least one in TSWV capture antibody (primary antibodie),
The IgG of usining contrasts as Quality Control.
Experimental example 1: see Fig. 1, a kind of antibody chip that is applied to detect feverfew virosis, 7 kinds of viral specificity capture antibodies (primary antibodie) of the harm feverfew that includes basement membrane and make respectively on basement membrane, described 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; On basement membrane, be also packaged with 3 positive quality control point P, point is made as the IgG of alkali phosphatase enzyme mark and is packaged with 3 negative Quality Control points, and point is made as the carbonate buffer solution of pH 9.2-9.8.。
The preparation of antibody chip:
(1) agents useful for same
Preparation capture antibody (primary antibodie) dilution buffer liquid (pH9.6): sodium carbonate Na
2cO
31.59g, sodium bicarbonate NaHCO
32.93g, sodium azide NaN
30.2g, distilled water ddH
2o1000mL, as chip sampling liquid;
Nitrocellulose filter: 0.22 μ m (article No.: 66485, U.S. PALL company).
(2) Dispersal risk chip
Antibody dilution: with capture antibody (primary antibodie) dilution buffer liquid dilution 7 kinds of polyclone capture antibodies (primary antibodie), BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV are followed successively by 1:17,1:14,1:17,1:14,1:15,1:3 and 1:15 times of antibody stoste for the antibody concentration of point sample, on chip, add the anti-goat IgG stoste of alkali phosphatase enzyme mark donkey of 1:10 dilution as the positive quality control point of chip, chip sampling liquid is as negative Quality Control point simultaneously;
Point sample: by after 0.22 μ m cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 ℃ of constant temperature ovens.Press chip lattice structure schematic diagram, see Fig. 1, every kind of polyclone capture antibody (primary antibodie) repeats 2 points of system; Respectively by for 7 kinds of viral capture antibodies (primary antibodie): anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus put the position at numbering 1-7 successively; Positive quality control, negative Quality Control are put respectively in position at P and N;
Mentioned reagent is pressed to array arrangement point sample in membrane surface, every some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in to fixedly 30min of 37 ℃, wet box (humidity is 45%).
Sealing: the antibody chip fixing is sealed to 1h without 37 ℃ of protein blocking liquid with 1% in wet box, remove after confining liquid, 2-8 ℃ of preservation, is antibody chip finished product.
Experimental example 2: a kind of kit that is applied to detect feverfew virosis, comprise antibody chip, the detected object of described antibody chip comprises BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV; The positive control of feverfew virus BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV; And the detection antibody (two is anti-) of the alkali phosphatase enzyme mark of feverfew virus BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV.
Chief component is as follows:
(1) the prepared antibody chip of experimental example 1 is 10;
(2) seven kinds of virus-positive testers, the detection antibody of the alkali phosphatase enzyme mark of specific binding plant virus (two is anti-) stoste and chromogenic substrate: 7 kinds of viruses described in experimental example 1 detect each 1 pipe of antibody (two is anti-) stoste accordingly; The marker enzyme that detects antibody (two is anti-) is alkaline phosphatase;
(3) detect antibody (two is anti-) dilution buffer liquid: pH=7.2-7.5, the phosphate buffer that Tween-20 mass content is 0.05%, BSA mass content is 0.2%, 1 bottle;
(4) lavation buffer solution PBST (sodium chloride nacl 8.0g, sodium hydrogen phosphate Na
2hPO
41.15g, potassium dihydrogen phosphate KH
2pO
40.2g, potassium chloride (KCl) 0.2g, Tween-200.5mL, distilled water 1000mL, pH=7.4), 1 bottle;
(5) the chloro-3-indyl of the bromo-4-of chromogenic substrate: 5-phosphate/NBT (BCIP/NBT) and 1 * alkaline phosphatase colour developing damping fluid, 1 cover.
Experimental example 3 antibody chips detect the specificity checking of target viral
By 7 kinds of viral positive controls, use respectively sample extraction damping fluid [polyvinylpyrrolidone (PVP, MW24000～40000) 20.0g, Na
2sO
31.3g, NaN
30.2g, ovalbumin 2.0g, Tween-2020.0mL, PBST1000mL, pH=7.4] dilution, the test sample liquid of preparation antibody chip, detects for antibody chip;
1) sample hybridization: by the test sample preparing, respectively with the antibody chip preparing overnight hatching at 4 ℃;
2) washing: PBST washes film with lavation buffer solution, totally 4 times, respectively washs 1min with 90r/min on horizontal shaking table;
3) detecting antibody (two is anti-) hatches: with pH=7.2-7.5, the phosphate buffer dilution that Tween-20 mass content is 0.05%, BSA mass content is 0.2% detect antibody (two is anti-) stoste to 1:600 doubly, preparation detects the mixed liquor of antibody (two is anti-) simultaneously containing 7 kinds of virus-specifics, be added on chip surface, on horizontal shaking table, with 30r/min, continue oscillating reactions 2-4h;
4) washing is with 2);
5) colour developing: the ratio preparation colour developing working fluid in BCIP:NBT:AP=1:1:20, be added on chip surface, under room temperature, react 20-30min; Diaphragm water after colour developing is completed rinses 3-5 time, makes it become the sampling point that atropurpureus can visual detection, then in 37 ℃ of dry 30min, after film is dry, with flat bed scanner, obtains detected image, then by sealed membrane sealing preservation for diaphragm.
Detect above-mentioned 7 kinds and viral the results are shown in Figure 2.1-7 is respectively the independent positive findings that detects BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV.Result demonstration, antibody chip has good specificity.
4 seven kinds of antiviral antibody chip detecting methods of experimental example are set up
Get 7 kinds of viral positive controls, with sample extraction damping fluid [polyvinylpyrrolidone (PVP, MW 24000～40000) 20.0g, Na
2sO
31.3g, NaN
30.2g, ovalbumin 2.0g, Tween-2020.0mL, PBST 1000mL, pH=7.4] dilution, the plant sample liquid of multiple virus is infected in random hybrid analog-digital simulation simultaneously.With ELISA method, the plant sample of each simulated infection is carried out to above-mentioned 7 kinds of viral detections, with antibody chip, detect simultaneously:
1) sample hybridization: by the test sample preparing, respectively with the antibody chip preparing overnight hatching at 4 ℃;
2) washing: PBST washes film with lavation buffer solution, totally 4 times, respectively washs 1min with 90r/min on horizontal shaking table;
3) detecting antibody (two is anti-) hatches: with pH=7.2-7.5, the phosphate buffer dilution that Tween-20 mass content is 0.05%, BSA mass content is 0.2% detect antibody (two is anti-) stoste to 1:600 doubly, preparation detects the mixed liquor of antibody (two is anti-) simultaneously containing 7 kinds of virus-specifics, be added on chip surface, on horizontal shaking table, with 30r/min, continue oscillating reactions 2-4h;
4) washing is with 2);
5) colour developing: the ratio preparation colour developing working fluid in BCIP:NBT:AP=1:1:20, be added on chip surface, under room temperature, react 20-30min; Diaphragm water after colour developing is completed rinses 3-5 time, makes it become atropurpureus sampling point that can visual detection, then in 37 ℃ of dry 30min, after film is dry, with flat bed scanner, obtains detected image, then by sealed membrane sealing preservation for diaphragm.
Testing result is shown in Fig. 3, for antibody chip method detects 7 kinds of viruses simultaneously, illustrates that antibody chip of the present invention has to detect above-mentioned 7 kinds of viral abilities simultaneously.With antibody chip, the sample accumulative total of 20 parts of simulated infections is filtered out to 41 infection altogether, basically identical with the testing result of ELISA method.
Experimental example 5 utilizes above-mentioned antibody chip to detect seed sample
Collect 50 parts of feverfew seed samples, virus entrained in seed is confirmed through serology and RT-PCR method.Adopt ELISA method to carry out respectively 7 kinds of viral detections to this 50 duplicate samples, apply antibody chip simultaneously these samples are detected, detecting step is with embodiment 3.Testing result demonstration, antibody chip method specificity is stronger, consistent with the testing result of ELISA method, and it not only has single viral detectability, but also has the ability that once experiment detects multiple virus simultaneously.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
1. an antibody chip that is applied to detect feverfew virosis, it is characterized in that, 7 kinds of viral specificity capture antibodies of the harm feverfew that includes basement membrane and make respectively on basement membrane, 7 kinds of described viral capture antibodies: anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; On basement membrane, be also packaged with 3 positive quality control point P, point is made as the IgG of alkali phosphatase enzyme mark and is packaged with 3 negative Quality Control point N, and point is made as the carbonate buffer solution of pH9.2-9.8.
2. the preparation method who is applied to detect the antibody chip of feverfew virosis as claimed in claim 1, is characterized in that step is:
(1) antibody dilution: with 7 kinds of polyclone capture antibodies of capture antibody dilution buffer liquid dilution, capture antibody concentration after dilution is 1/18-1/3 times of antibody stoste, it is BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV are followed successively by antibody stoste for the antibody concentration of point sample 1:17,1:14,1:17,1:14,1:15,1:3 and 1:15 times, on chip, add the anti-goat IgG stoste of alkali phosphatase enzyme mark donkey of 1:10 dilution as the positive quality control point of chip, chip sampling liquid is as negative Quality Control point simultaneously; Point sample damping fluid is: sodium carbonate Na
2cO
31.59g, sodium bicarbonate NaHCO
32.93g, sodium azide NaN
30.2g, distilled water ddH
2o 1000mL, pH 9.6;
(2) point sample: by after cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 ℃ of constant temperature ovens.Press chip dot matrix, every kind of polyclone capture antibody repeats 2 points of system; Respectively for 7 kinds of viral capture antibodies: anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus put the position at numbering 1-7 successively; Positive quality control, negative Quality Control are put respectively in position at P and N;
(3) mentioned reagent is pressed to array arrangement point sample in membrane surface, every some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in to wet box, humidity is 45%, 37 ℃ of fixedly 30min;
(4) sealing: the antibody chip fixing is sealed to 1h without 37 ℃ of protein blocking liquid with 1% in wet box, remove after confining liquid, 2-8 ℃ of preservation, is antibody chip finished product.
3. an antibody chip kit that is applied to detect feverfew virosis, is characterized in that, includes antibody chip; The detection antibody of the alkali phosphatase enzyme mark that 7 kinds of virus is corresponding: anti-bean virus 2, anti-arabis mosaic virus, anti-bean bacterial northern wilt poison, anti-cucumber mosaic virus, anti-Tobacco rattle virus, anti-nepovirus, anti-tomato spotted wilf virus; The positive control of feverfew virus, include the positive control of bean virus 2, the positive control of arabis mosaic virus, the positive control of bean bacterial northern wilt poison, the positive control of the positive control of cucumber mosaic virus, Tobacco rattle virus, the positive control of the positive control of nepovirus, tomato spotted wilf virus; Alkaline phosphatase; Detect antibody dilution buffer; Lavation buffer solution PBST; The chloro-3-indyl of the bromo-4-of alkaline phosphatase chromogenic substrate 5-phosphate/NBT.
4. a detection method that is applied to detect the antibody chip kit of feverfew virosis, is characterized in that step is:
(1) by detection sample and 4 ℃ of overnight hatching of antibody chip; Described antibody chip is claim 1;
(2) after washing, add again corresponding plant virus to detect antibody hybridization and hatch, continue reaction 4h;
(3) after washing, add the colour developing of colour developing working fluid;
Described feverfew virus capture antibody is respectively bean virus 2 Bean yellow mosaic virus, BYMV, arabis mosaic virus Arabis mosaic virus, ArMV, bean bacterial northern wilt poison Broad bean wilt virus, BBWV, cucumber mosaic virus Cucumber mosaic virus, CMV, Tobacco rattle virus Tobacco rattle virus, TRV, nepovirus Tobacco ringspot virus, TRSV and tomato spotted wilf virus Tomato spotted wilt virus, at least one in TSWV capture antibody;
The IgG of usining contrasts as Quality Control.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
| CN112898422A (en) * | 2021-04-12 | 2021-06-04 | 中国计量大学 | Tobacco ringspot virus monoclonal antibody and preparation method and application thereof |
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| CN1996020A (en) * | 2005-12-31 | 2007-07-11 | 中国科学院寒区旱区环境与工程研究所 | Double-antibody sandwich enzyme-linked immunologic adsorption detection kit for cucumber mosaic virus and method for preparing same |
| KR20120088917A (en) * | 2011-02-01 | 2012-08-09 | 중앙대학교 산학협력단 | Detecting method of CMV or GMO that tolerant to CMV |
| CN102735836A (en) * | 2011-04-01 | 2012-10-17 | 上海慧耘生物科技有限公司 | Visual rapid combined measuring method of plant pathogen and antibody chip |
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Patent Citations (4)
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| EP0268465A2 (en) * | 1986-11-19 | 1988-05-25 | Bioclones (Proprietary) Limited | Method for detecting the presence of a plant antigen in a plant |
| CN1996020A (en) * | 2005-12-31 | 2007-07-11 | 中国科学院寒区旱区环境与工程研究所 | Double-antibody sandwich enzyme-linked immunologic adsorption detection kit for cucumber mosaic virus and method for preparing same |
| KR20120088917A (en) * | 2011-02-01 | 2012-08-09 | 중앙대학교 산학협력단 | Detecting method of CMV or GMO that tolerant to CMV |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
| CN112898422A (en) * | 2021-04-12 | 2021-06-04 | 中国计量大学 | Tobacco ringspot virus monoclonal antibody and preparation method and application thereof |
| CN112898422B (en) * | 2021-04-12 | 2022-08-30 | 中国计量大学 | Tobacco ringspot virus monoclonal antibody and preparation method and application thereof |
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| CN104111331B (en) | 2015-05-27 |
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