CN104111331B - 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 and be a kind ofly applied to the examination of the viroses of plant and the antibody chip of diagnosis and kit and method thereof, mainly for the bean virus 2 of feverfew, arabis mosaic virus, bean bacterial northern wilt poison, cucumber mosaic virus, Tobacco rattle virus, nepovirus and tomato spotted wilf virus 7 kinds of viruses, belong to biological technical field.
Background technology
Have medicinal in a large number in feverfew, 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 mosaicvirus, ArMV), bean bacterial northern wilt poison (Broad bean wilt virus, BBWV), cucumber mosaic virus (Cucumbermosaic virus, CMV), Tobacco rattle virus (Tobacco rattle virus, TRV), nepovirus (Tobaccoringspot virus, and tomato spotted wilf virus (Tomato spotted wilt virus TRSV), TSWV) all feverfew can be infected.BYMV infects the various plants such as composite family, Chenopodiaceae, Amaranthaceae, Aizoaceae; This viral communication media mainly aphid, propagates with non-persistent manner, also can propagate by mechanical inoculation.ArMV is that China enters the territory the quarantine harmful organisms of plant, can endanger about 174 and belong to 200 various plants, comprise composite vegetable, flowers etc., cause the symptoms such as floral leaf, yellow chlorisis, dwarfing, shrinkage, necrosis; This virus is propagated by the approach such as nematode and seed.BBWV host range is wide, can infect 328 Plants in 44 sections 186 genus, comprise multiple feverfew; The symptom such as cause floral leaf, dwarfing, wilting and plant withered; Occurring in nature is propagated with non-persistent manner by multiple aphid, and The book of Changes mechanical inoculation is propagated.CMV is worldwide distributing virus, can infect more than 1000 kind of unifacial leaf and dicotyledon, is that host plant is maximum, distribution is the widest, the plant virus of most Economic Importance; CMV mainly relies on multiple aphid for passing malicious medium, and propagate with non-persistent manner, easily propagated by mechanical inoculation, the seed of Chinese dodder also can pass poison.TRV host range is wide, can infect 50 section, 400 kinds of dicotyledonous and monocotyledons, can infect the vegetables and flowers such as sunflower, lettuce under natural conditions; Occurring in nature is mainly through intending burr nematode and the propagation of burr nematode, and water, seed or the seed of Chinese dodder also can pass poison.TRSV is the quarantine harmful organisms can propagated with vegetable seeds, nursery stock, and can cause harm 54 section 300 various plants, and natural infection host comprises multiple composite vegetable, flowers etc.; The Symptoms infected by TRSV is different because of host, and ring-type or wire line, chlorisis spot, necrotic plaque etc. appear in usual blade; 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 that China enters the territory plant quarantine harmful organism, 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, is easily propagated with persistent fashion by thrips, and grafting also can pass poison.
Feverfew infects the multiple symptom of rear performance by virosis, as floral leaf, deformity, yellow, stunt, downright bad etc., diseased plant depauperation, causes yield and quality greatly to reduce.In field, after feverfew infects virosis, its symptom affects very large by kind, plant growing stage, environmental baseline and virus strain, therefore be difficult to judge virosis by any or which kind virus is caused actually from Symptoms, therefore the synchronization detecting method of high flux, highly sensitive multiple feverfew virus, for finding out cause of disease in time, eliminating primary source of infection, preventing its spread in china of virosis most important.Current application more widely plant virus detection method has enzyme linked immunosorbent assay (ELISA) to test (Enzyme Linked ImmunoSorbent Assay, and reverse transcription polymerase chain amplification (Reverse Transcription-PCR ELISA), RT-PCR), but adopt the multiple cause of diseases in conventional method detection sample, process is very long, needs to spend a large amount of human and material resources and financial resources.
Antibody chip is that utilize between antigen and antibody molecule can the principle of specific binding, by capture antibody with the arrangement of array pattern point sample, after analyzed example reaction, obtains hybridization signal, finally determines distribution and the content information of target molecule in sample.Antibody chip has can carry out high flux parallel analysis, save reagent and material, the simple feature of sample handling processes, has been applied to multiple 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 being applied to and detecting feverfew virosis be provided, the described this antibody chip for Combining diagnosis 7 kinds of feverfew viruses have easy, quick, sensitive, specificity is good, the feature of flux.
An also object of the present invention is that providing a kind of is applied to the antibody chip kit detecting feverfew virosis.
Another object of the present invention is a kind of antibody chip detection method being applied to detection feverfew virosis.
For achieving the above object, the technical scheme that the present invention takes is: a kind of antibody chip being applied to detection feverfew virosis, it is characterized in that, include basement membrane and on basement membrane, put the viral specificity capture antibody (primary antibodie) of 7 kinds of harm feverfew of system respectively, described 7 kinds 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; Basement membrane is also packaged with 3 positive quality control point P, and 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 being applied to the antibody chip detecting feverfew virosis, the steps include:
(1) antibody dilution: dilute 7 kinds of polyclone capture antibodies (primary antibodie) with capture antibody (primary antibodie) dilution buffer, capture antibody (primary antibodie) concentration after dilution is 1/18-1/3 times of antibody stoste, namely 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 of antibody stoste doubly for the antibody concentration of point sample, chip adds the positive quality control point of alkali phosphatase enzyme mark donkey anti goat igg stoste as chip of 1:10 dilution, chip sampling liquid is as negative Quality Control point simultaneously; Spotting buffer 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: after cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 DEG C of constant temperature ovens.By chip dot matrix, often kind of polyclone capture antibody (primary antibodie) repeats system 2 points; 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 is put respectively in the position of P and N;
(3) mentioned reagent is pressed array arrangement point sample in membrane surface, often some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in wet box, humidity is 45%, 37 DEG C of fixing 30min;
(4) close: the antibody chip fixed is closed 1h without protein blocking liquid 37 DEG C with 1% in wet box, after removing confining liquid, 2-8 DEG C of preservation, is antibody chip finished product.
Be applied to the antibody chip kit detecting feverfew virosis, its principal feature is, includes antibody chip; The detection antibody (two resist) 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, includes 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 cucumber mosaic virus, the positive control of Tobacco rattle virus, the positive control of nepovirus, the positive control of tomato spotted wilf virus; Alkaline phosphatase; Detect antibody (two resist) dilution buffer; Lavation buffer solution PBST; The chloro-3-indolyl phosphate/NBT of the bromo-4-of alkaline phosphatase chromogenic substrate 5-.
Be applied to a detection method for the antibody chip kit detecting feverfew virosis, its principal feature is that step is:
(1) by detection sample and antibody chip 4 DEG C is overnight hatches; Described antibody chip is the viral specificity capture antibodies (primary antibodie) of 7 kinds of the harm feverfew including basement membrane and put system respectively on basement membrane, described 7 kinds 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; Basement membrane is also packaged with 3 positive quality control point P, and 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) add corresponding plant virus after washing again and detect antibody (two resist) hybridization incubation, continue reaction 4h;
(3) colour developing of colour developing working fluid is added after washing;
Described feverfew virus capture antibody (primary antibodie) is respectively bean virus 2 Bean yellow mosaicvirus, BYMV, arabis mosaic virus Arabis mosaic virus, ArMV, bean bacterial northern wilt poison Broad bean wiltvirus, BBWV, cucumber mosaic virus Cucumber mosaic virus, CMV, Tobacco rattle virus Tobacco rattlevirus, TRV, nepovirus Tobacco ringspot virus, TRSV and tomato spotted wilf virus Tomato spottedwilt virus, at least one in TSWV capture antibody (primary antibodie),
Contrast using IgG as Quality Control.
Principle of the present invention is: choose respectively for the specificity capture antibody (primary antibodie) of these 7 kinds of cause of diseases, be fixed on cellulose nitrate basement membrane with array pattern, utilize cause of disease in capture antibody (primary antibodie) specific binding sample, react with the detection antibody (two resist) of alkali phosphatase enzyme mark again, and then the double-antibody sandwich structure of " capture antibody-antigen-detection antibody " is formed in array surface specificity, utilize the specific chromogenic substrate display hybridization signal that alkaline phosphatase is corresponding subsequently, 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 corresponding positive control and the polyclone capture antibody (primary antibodie) that detect virus, 7 identical basement membranes add mixing positive control, and hybrid detection antibody (two resist), have no obvious non-specific cross-reaction, establish sensing range, lowest detectable limit, the precision of double antibody sandwich method to each virus, successfully construct the multiple antibody chip detection system that can be used for sample examination.With seed and plant leaf blade extract hot-wire array to the detectability of actual sample, result shows its positive rate and to be better than or suitable with ELISA, demonstrates good practical prospect.
Beneficial effect of the present invention: the present invention successfully constructs the antibody chip detecting the multiple virus of feverfew, comprehensive detection can be carried out to multiple cause of disease 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, and operation is convenient, and cost is low; (2) detection to multiple sample to be checked can be realized simultaneously, as many as 7 Plants virus can be detected in once testing simultaneously, improve the diagnosis efficiency of virosis, save human and material resources and financial resources; (3) detect sample preparation simple, 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 the sensitivity equal with methods such as ELISA, specificity, 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 the testing result display image of embodiment 2 antibody chip specificity verification experiment.
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 chip detects 7 kinds of viral the results simultaneously.
Embodiment
Be described principle of the present invention and feature below in conjunction with embodiment, 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 being applied to detection feverfew virosis, it is characterized in that, include basement membrane and on basement membrane, put the viral specificity capture antibody (primary antibodie) of 7 kinds of harm feverfew of system respectively, described 7 kinds 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; Basement membrane is also packaged with 3 positive quality control point P, and 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: the described preparation method being applied to the antibody chip detecting feverfew virosis, the steps include:
(1) antibody dilution: dilute 7 kinds of polyclone capture antibodies (primary antibodie) with capture antibody (primary antibodie) dilution buffer, capture antibody (primary antibodie) concentration after dilution is 1/18-1/3 times of antibody stoste, namely 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 of antibody stoste doubly for the antibody concentration of point sample, chip adds the positive quality control point of alkali phosphatase enzyme mark donkey anti goat igg stoste as chip of 1:10 dilution, chip sampling liquid is as negative Quality Control point simultaneously; Spotting buffer 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: after cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 DEG C of constant temperature ovens.By chip dot matrix, often kind of polyclone capture antibody (primary antibodie) repeats system 2 points; 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 is put respectively in the position of P and N;
(3) mentioned reagent is pressed array arrangement point sample in membrane surface, often some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in wet box, humidity is 45%, 37 DEG C of fixing 30min;
(4) close: the antibody chip fixed is closed 1h without protein blocking liquid 37 DEG C with 1% in wet box, removing confining liquid, 2-8 DEG C of preservation, is antibody chip finished product.
Embodiment 3: a kind of antibody chip kit being applied to detection feverfew virosis, its principal feature is, includes antibody chip; The detection antibody (two resist) 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, includes 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 cucumber mosaic virus, the positive control of Tobacco rattle virus, the positive control of nepovirus, the positive control of tomato spotted wilf virus; Alkaline phosphatase; Detect antibody (two resist) dilution buffer; Lavation buffer solution PBST; The chloro-3-indolyl phosphate/NBT of the bromo-4-of alkaline phosphatase chromogenic substrate 5-.
Embodiment 4: a kind of detection method being applied to the antibody chip kit detecting feverfew virosis, its principal feature is that step is:
(1) by detection sample and antibody chip 4 DEG C is overnight hatches; Described antibody chip is the specificity capture antibody (primary antibodie) of the virus including basement membrane and put 7 kinds of system harm feverfews respectively on basement membrane, described 7 kinds 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; Basement membrane is also packaged with 3 positive quality control point P, and 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) add corresponding plant virus after washing again and detect antibody (two resist) hybridization incubation, continue reaction 4h;
(3) colour developing of colour developing working fluid is added after washing:
Described feverfew virus capture antibody (primary antibodie) is respectively bean virus 2 Bean yellow mosaicvirus, BYMV, arabis mosaic virus Arabis mosaic virus, ArMV, bean bacterial northern wilt poison Broad bean wiltvirus, BBWV, cucumber mosaic virus Cucumber mosaic virus, CMV, Tobacco rattle virus Tobacco rattlevirus, TRV, nepovirus Tobacco ringspot virus, TRSV and tomato spotted wilf virus Tomato spottedwilt virus, at least one in TSWV capture antibody (primary antibodie),
Contrast using IgG as Quality Control.
Experimental example 1: see Fig. 1, a kind of antibody chip being applied to detection feverfew virosis, include basement membrane and on basement membrane, put the viral specificity capture antibody (primary antibodie) of 7 kinds of harm feverfew of system respectively, described 7 kinds 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; Basement membrane is also packaged with 3 positive quality control point P, and 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 (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 μm (article No.: 66485, PALL company of the U.S.).
(2) Dispersal risk chip
Antibody dilution: dilute 7 kinds of polyclone capture antibodies (primary antibodie) with capture antibody (primary antibodie) dilution buffer, the antibody concentration that BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV are used for point sample is followed successively by 1:17,1:14,1:17,1:14,1:15,1:3 and 1:15 of antibody stoste doubly, chip adds the positive quality control point of alkali phosphatase enzyme mark donkey anti goat igg stoste as chip of 1:10 dilution, chip sampling liquid is as negative Quality Control point simultaneously;
Point sample: after 0.22 μm of cellulose nitrate diaphragm trace, deionized water soaks 20min, dry 20min in 37 DEG C of constant temperature ovens.By chip lattice structure schematic diagram, see Fig. 1, often kind of polyclone capture antibody (primary antibodie) repeats system 2 points; 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 is put respectively in the position of P and N;
Mentioned reagent is pressed array arrangement point sample in membrane surface, often some 0.3uL, dot spacing 0.2cm.After point sample, antibody chip is placed in wet box (humidity is 45%) 37 DEG C of fixing 30min.
Close: the antibody chip fixed is closed 1h without protein blocking liquid 37 DEG C with 1% in wet box, after removing confining liquid, 2-8 DEG C of preservation, is antibody chip finished product.
Experimental example 2: a kind of kit being applied to detection 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 resist) of the alkali phosphatase enzyme mark of feverfew virus BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV.
Mainly composed as follows:
(1) antibody chip prepared by experimental example 1 10;
(2) seven kinds of virus positive control things, detection antibody (two resist) stoste of the alkali phosphatase enzyme mark of specific binding plant virus and chromogenic substrate: 7 kinds of viruses described in experimental example 1 detect each 1 pipe of antibody (two resist) stoste accordingly; The marker enzyme detecting antibody (two resist) is alkaline phosphatase;
(3) detect antibody (two resist) dilution buffer: pH=7.2-7.5, Tween-20 mass content is 0.05%, BSA mass content is the phosphate buffer of 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-indolyl phosphate/NBT (BCIP/NBT) of the bromo-4-of chromogenic substrate: 5-and 1 × alkaline phosphatase colorbuffer, 1 cover.
Experimental example 3 antibody chip detects the specificity verification of target viral
By 7 kinds of viral positive controls, use sample extraction damping fluid [polyvinylpyrrolidone (PVP, MW24000 ~ 40000) 20.0g, Na respectively
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 prepared, overnightly at 4 DEG C with the antibody chip prepared respectively hatches;
2) wash: wash film with lavation buffer solution PBST, totally 4 times, horizontal shaker respectively washs 1min with 90r/min;
3) detect antibody (two resist) to hatch: with pH=7.2-7.5, the phosphate buffer dilution that Tween-20 mass content is 0.05%, BSA mass content is 0.2% detects antibody (two resist) stoste to 1:600 times, preparation detects the mixed liquor of antibody (two resist) simultaneously containing 7 kinds of virus-specifics, be added on chip surface, horizontal shaker continues oscillating reactions 2-4h with 30r/min;
4) washing is with 2);
5) develop the color: by the proportions colour developing working fluid of BCIP:NBT:AP=1:1:20, be added on chip surface, under room temperature, react 20-30min; Rinsed 3-5 time by diaphragm water after having developed the color, making it become atropurpureus can the sampling point of visual detection, then in 37 DEG C of dry 30min, after film drying, obtains detected image with flat bed scanner, then by diaphragm sealed membrane sealing preservation.
Detect above-mentioned 7 kinds and viral the results are shown in Figure 2.1-7 is respectively the positive findings detecting separately BYMV, ArMV, BBWV, CMV, TRV, TRSV and TSWV.Result shows, and antibody chip has good specificity.
Experimental example 4 seven kinds of antiviral antibody chip detecting methods 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, above-mentioned 7 kinds of viral detections are carried out to the plant sample of each simulated infection, detect with antibody chip simultaneously:
1) sample hybridization: by the test sample prepared, overnightly at 4 DEG C with the antibody chip prepared respectively hatches;
2) wash: wash film with lavation buffer solution PBST, totally 4 times, horizontal shaker respectively washs 1min with 90r/min;
3) detect antibody (two resist) to hatch: with pH=7.2-7.5, the phosphate buffer dilution that Tween-20 mass content is 0.05%, BSA mass content is 0.2% detects antibody (two resist) stoste to 1:600 times, preparation detects the mixed liquor of antibody (two resist) simultaneously containing 7 kinds of virus-specifics, be added on chip surface, horizontal shaker continues oscillating reactions 2-4h with 30r/min;
4) washing is with 2);
5) develop the color: by the proportions colour developing working fluid of BCIP:NBT:AP=1:1:20, be added on chip surface, under room temperature, react 20-30min; Diaphragm water after having developed the color is rinsed 3-5 time, make its become atropurpureus can the sampling point of visual detection, then in 37 DEG C of dry 30min, after film drying, obtain detected image with flat bed scanner, then by diaphragm sealed membrane sealing preservation.
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 and detects above-mentioned 7 kinds of viral abilities simultaneously.41 infection are filtered out altogether to the sample of 20 parts of simulated infections is accumulative with antibody chip, 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 confirms through serology and RT-PCR method.Adopt ELISA method to carry out 7 kinds of viral detections respectively to these 50 increment product, apply antibody chip simultaneously and detect these samples, detecting step is with embodiment 3.Testing result shows, and antibody chip method specificity is comparatively strong, and consistent with the testing result of ELISA method, it not only has single Viral diagnosis ability, but also has the ability of once testing and detecting multiple virus simultaneously.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. one kind is applied to the antibody chip detecting feverfew virosis, it is characterized in that, include basement membrane and on basement membrane, put the viral specificity capture antibody of 7 kinds of harm feverfew of system respectively, described 7 kinds 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; Basement membrane is also packaged with 3 positive quality control point P, and 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;
The described preparation method being applied to the antibody chip detecting feverfew virosis is:
(1) antibody dilution: dilute 7 kinds of polyclone capture antibodies with capture antibody dilution buffer, capture antibody concentration after dilution is 1/18-1/3 times of antibody stoste, namely 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 of antibody stoste doubly for the antibody concentration of point sample, chip adds the positive quality control point of alkali phosphatase enzyme mark donkey anti goat igg stoste as chip of 1:10 dilution, chip sampling liquid is as negative Quality Control point simultaneously; Spotting buffer 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: after cellulose nitrate diaphragm trace, deionized water soaks 20 min, dry 20 min in 37 DEG C of constant temperature ovens; By chip dot matrix, often kind of polyclone capture antibody repeats system 2 points; 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 is put respectively in the position of P and N;
(3) mentioned reagent is pressed array arrangement point sample in membrane surface, often some 0.3uL, dot spacing 0.2cm; After point sample, antibody chip is placed in wet box, humidity is 45%, 37 DEG C of fixing 30min;
(4) close: the antibody chip fixed is closed 1h without protein blocking liquid 37 DEG C with 1% in wet box, after removing confining liquid, 2-8 DEG C of preservation, is antibody chip finished product.
2. be applied to the kit of the antibody chip detecting feverfew virosis as claimed in claim 1, it is characterized in that, include 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, includes 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 cucumber mosaic virus, the positive control of Tobacco rattle virus, the positive control of nepovirus, the positive control of tomato spotted wilf virus; Alkaline phosphatase; Detect antibody dilution buffer; Lavation buffer solution PBST; The chloro-3-indolyl phosphate/NBT of the bromo-4-of alkaline phosphatase chromogenic substrate 5-.
3. be applied to the detection method of the antibody chip kit detecting feverfew virosis as claimed in claim 2, it is characterized in that step is:
(1) by detection sample and antibody chip 4 DEG C is overnight hatches; Described antibody chip is claim 1;
(2) add corresponding plant virus detection antibody hybridization after washing again to hatch, continue reaction 4h;
(3) colour developing of colour developing working fluid is added after washing;
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;
Contrast using IgG as Quality Control.
4. be applied to a preparation method for the antibody chip detecting feverfew virosis, it is characterized in that, comprise the steps:
(1) antibody dilution: dilute 7 kinds of polyclone capture antibodies with capture antibody dilution buffer, capture antibody concentration after dilution is 1/18-1/3 times of antibody stoste, namely 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 of antibody stoste doubly for the antibody concentration of point sample, chip adds the positive quality control point of alkali phosphatase enzyme mark donkey anti goat igg stoste as chip of 1:10 dilution, chip sampling liquid is as negative Quality Control point simultaneously; Spotting buffer 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: after cellulose nitrate diaphragm trace, deionized water soaks 20 min, dry 20 min in 37 DEG C of constant temperature ovens; By chip dot matrix, often kind of polyclone capture antibody repeats system 2 points; 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 is put respectively in the position of P and N;
(3) mentioned reagent is pressed array arrangement point sample in membrane surface, often some 0.3uL, dot spacing 0.2cm; After point sample, antibody chip is placed in wet box, humidity is 45%, 37 DEG C of fixing 30min;
(4) close: the antibody chip fixed is closed 1h without protein blocking liquid 37 DEG C with 1% in wet box, after removing confining liquid, 2-8 DEG C of preservation, is antibody chip finished product.
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| CN201410382666.8A CN104111331B (en) | 2014-08-06 | 2014-08-06 | Antibody chip for detecting compositae plant virus diseases, and kit and method of antibody chip |
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| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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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