CN102043056A - Preparation method for immune colloidal gold test strip for simultaneously detecting orchid viruses CyMV and ORSV - Google Patents
Preparation method for immune colloidal gold test strip for simultaneously detecting orchid viruses CyMV and ORSV Download PDFInfo
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Abstract
The invention discloses a preparation method for an immune colloidal gold test strip for simultaneously detecting orchid viruses CyMV and ORSV. The preparation method comprises the following steps of: dipping CyMV and ORSV monoclonal antibody solution on a detection line of a nitrocellulose membrane respectively in turn; dipping a goat-anti-rabbit polyclonal antibody on a quality control line of the nitrocellulose membrane; and respectively pasting a sample pad prepared by absorbent filter paper, a CyMV and ORSV immune colloidal gold pad, the nitrocellulose membrane and absorbent paper to a PVC slab rubber with a non-setting adhesive in turn to form the test strip. The preparation method has the advantages that: the preparation method is simple and fast to operate, the result is accurate and the cost is low; the pollution on an orchid sample caused by sampling repeatedly is reduced; and unnecessary consumptive material waste is also reduced.
Description
Technical field
The present invention relates to a kind of preparation method of immunity colloidal gold test paper strip, particularly a kind of preparation method that can detect orchid virus CyMV and ORSV immunity colloidal gold test paper strip simultaneously.
Background technology
Orchid becomes most popular kind in the potted flower because characteristics such as it is wide in variety, pattern is gorgeous, the florescence is long are liked by the common people deeply, and in recent years, orchid was soon at the speed of development of China, head and shoulders above people's expectation.But the orchid and the viral disease that easily catches a disease, virus causes the whole body systemic infection on the orchid plant, make blue strain poor growth, and blade the mosaic of striped, patch, gangrene or green skewness occurs and levies.Any propagation that can cause the chance of mechanicalness wound, the basin alms bowl of cultivating diseased plant even irrigation water all can cause virus of being taked during the artificial cultivation iris; add that in recent years orchid industry is gradually after the scale; in order to reach the tissue culture technology that quick breeding seedling is adopted, more quickened comprehensively spreading of virus.
In various known orchid diseases, spread to offspring's seedling with asexual mitogenetic breeding especially easily with virosis (Virusdiseases).The viral species that can infect orchid has 29 kinds at least.But what generally take place has only cymbidium mosaic virus (Cymbidium mosaic virus, abbreviation CymMV), the blue zonate spot poison of tooth (Odontoglossumringspot virus, be called for short ORSV) and three kinds of cucumber mosaic virus (Cucumber mosaic virus is called for short CMV) etc.But multinomial survey report shows that infection conditions is the most general, to global orchid industry influence the most serious be CyMV and ORSV.According to estimates, the iris seedling that current China producer uses almost carries this two kinds of viruses more than 90%, and in the iris seedling that the Taiwan produces, the carrying rate of these two kinds of viruses is also about 70%.Therefore can effectively improve the detection efficiency of orchid virus, not only can improve the economic benefit of orchid seedling, also can improve the competitive power of domestic orchid industry.
At present the detection method of orchid virus is mainly comprised the research of phyto-indicator method, electron microscopic observation method, serological method and molecular detection technology, comprise RT-PCR, molecular probe etc.
1. phyto-indicator method:, exist a lot of not enough though the phyto-indicator method is simple.Mostly be in reality is identified that several viruses are compound to be infected, the symptom performance changes bigger, and the performance of the symptom on the different times phyto-indicator is also variant, thereby has increased detection difficulty; Detection speed is slow, and is periodically long, need time 3-20 not wait in several days, and sensitivity is lower; The performance of postvaccinal symptom is subjected to the influence of the restriction in season and external environment bigger, safeguards that phyto-indicator garden expense is also high.
2. electron microscopic observation method: this method is simple and efficient, be applicable to the detection of a large amount of samples, but because electron microscopy needs the electron microscope of higher multiple, and the preparation expense of sample is higher, it is bigger to extract viral difficulty, so Electron Microscopy is subjected to certain restriction.
3. serological method: utilize the immunological response of antigen-antibody to detect whether viruliferous method of plant.Mainly be to adopt enzyme linked immunosorbent assay (ELISA) and some immunity at present, exist following shortcoming: a only to be applicable to and the detection of a large amount of samples can not realize the detection of simple sample in conjunction with test (DIBA); B needs the technical operation personnel of special instrument and equipment and specialty, is unfavorable for promoting; C operating process relative complex, detection time is long, does not wait in 4-12 hour.Now also developed orchid virus gold-immunochromatographyreagent reagent for assay paper, this is a kind of of serological method, is newer a kind of method during orchid detects in recent years.Because can only carry out the single detection of virus, cost is higher, do not obtain promoting.
4. molecular detection technology: Protocols in Molecular Biology commonly used at present comprises making nucleic acid molecular hybridization technology and RT-PCR method.RT-PCR method detect plant virus have sensitivity, fast, advantage such as high specificity, its selectivity and sensitivity then are better than immunological method, and the multiple detection of having developed two kinds of viruses at present.But because orchid virus is a RNA viruses, RNA is easy to degraded, and extraction and operating process are higher for environment and technical requirement, needs expensive reagent and special equipment during experiment, can only be unfavorable for popularizing carrying out in the laboratory preferably.
5. Lin Zhikeng, the Wu Zujian etc. of domestic 2007 University Of Agriculture and Forestry In Fujian once invented the viral test strip of single kind that is applied to orchid virus, though it adopts identical principle, but the polyclonal antibody that it adopts, a little less than sensitivity is wanted than monoclonal antibody, specificity is relatively poor, can not detect two kinds of viruses simultaneously simultaneously, needs separate detection, whole length consuming time, and waste consumptive material.
Summary of the invention
The objective of the invention is to provide a kind of preparation method that can detect the immune colloid gold test paper of orchid virus CyMV and ORSV simultaneously, this test paper can disposable fast detecting CyMV and two kinds of viruses of ORSV, reduce and repeatedly sample, also can reduce unnecessary consumptive material waste the pollution of orchid sample.
The present invention is achieved in that the described preparation method who detects orchid virus CyMV and ORSV immunity colloidal gold test paper strip simultaneously, comprises the steps:
A) CyMV MONOCLONAL ANTIBODIES SPECIFIC FOR: obtain the CyMV virus coat protein gene by the homologous clone method, make up prokaryotic expression carrier, obtain the external source fusion of CyMV-CP by abduction delivering, perhaps that expression and purification is good external source fusion injection mouse, the preparation fused cell, and by screening, the high monoclonal antibody of preparation specificity, purification antibody, purity is not less than 95%;
B) ORSV MONOCLONAL ANTIBODIES SPECIFIC FOR: obtain the ORSV virus coat protein gene by the homologous clone method, make up prokaryotic expression carrier, obtain the external source fusion of ORSV-CP by abduction delivering, perhaps that expression and purification is good external source fusion injection mouse, the preparation fused cell, and by screening, the high monoclonal antibody of preparation specificity, purification antibody, purity is not less than 95%;
C) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-60nm with 1% trisodium citrate of 2-3ml;
D) collaurum redissolves liquid: 1% bovine serum albumin(BSA), 0.5% degreasing dry powder, 0.05% sodium azide NaN3,1% Tween-20, the 0.01MpH7.0 phosphate buffer, the 0.22U membrane filtration, put 2-8 ℃ standby, the term of validity 15 days;
E) CyMV immune colloid gold solution: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg CyMV monoclonal antibody in the 100ml colloidal gold solution, mix, and the adding bovine serum albumin(BSA) seals, making its final concentration is 0.5%-2%, the centrifugal supernatant that goes, the collaurum redissolution liquid of adding 1/10th volumes obtains CyMV immune colloid gold solution;
F) ORSV immune colloid gold solution: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg ORSV monoclonal antibody in the 100ml colloidal gold solution, mix, and the adding bovine serum albumin(BSA) seals, making its final concentration is 0.5%-2%, the centrifugal supernatant that goes, the collaurum redissolution liquid of adding 1/10th volumes obtains ORSV immune colloid gold solution;
G) with e) the CyMV immune colloid gold solution and the f of step) the ORSV immune colloid gold solution of step mixes and adds a certain amount of collaurum and redissolve liquid, until under electron microscope, observing CyMV and ORSV immune colloid gold solution evenly mixes, above-mentioned mixed solution evenly is layered on 22-50cm for every milliliter
2Glass fibre on, dry back forms CyMV and ORSV immune colloid gold pad;
H) CyMV and ORSV monoclonal anti liquid solution are put on the detection line of nitrocellulose membrane respectively in order, goat-anti rabbit polyclonal antibody point is on the nature controlling line of nitrocellulose membrane;
I) will paste successively by sample pad, CyMV and ORSV immune colloid gold pad, nitrocellulose filter and the thieving paper that absorbent filter is made respectively on the PVC offset plate of a band adhesive sticker, become test strips; And on test strips, be stained with and have the transparent non-setting adhesive that inserts the highest spacing index line MAX of liquid levels;
J) test strips of drying is packed preservation with foil sealing.
The invention has the beneficial effects as follows, the used immunizing antigen of the present invention is a prokaryotic expression antigen, is at special virus capsid protein design and the antigen of expressing, its immunogenicity height, prepared antibody capable specific recognition virus capsid protein, thus sensitivity and the accuracy that detects improved; Detect orchid virus with the present invention, detection time is short, and it is low to detect cost, the accuracy of detection height.
Description of drawings
Fig. 1 is a synoptic diagram of the present invention.
Fig. 2,3 is the template drawing on the present invention's packing.
Among the figure: 1. the sample pad made of absorbent filter, 2. CyMV and ORSV immune colloid gold pad, 3. nitrocellulose filter, 4. thieving paper, 5. PVC offset plate, 6. CyMV monoclonal anti liquid solution point is at detection line, and 7. ORSV monoclonal anti liquid solution point is at detection line, and 8. goat-anti rabbit polyclonal antibody point is at nature controlling line.
Embodiment
The preparation method who detects orchid virus CyMV and ORSV immunity colloidal gold test paper strip simultaneously of the present invention as shown in Figure 1, 2, 3, comprises the steps:
A) CyMV MONOCLONAL ANTIBODIES SPECIFIC FOR: obtain the CyMV virus coat protein gene by the homologous clone method, make up prokaryotic expression carrier, obtain the external source fusion of CyMV-CP by abduction delivering, perhaps that expression and purification is good external source fusion injection mouse, the preparation fused cell, and by screening, the high monoclonal antibody of preparation specificity, purification antibody, purity is not less than 95%;
B) ORSV MONOCLONAL ANTIBODIES SPECIFIC FOR: obtain the ORSV virus coat protein gene by the homologous clone method, make up prokaryotic expression carrier, obtain the external source fusion of ORSV-CP by abduction delivering, perhaps that expression and purification is good external source fusion injection mouse, the preparation fused cell, and by screening, the high monoclonal antibody of preparation specificity, purification antibody, purity is not less than 95%;
C) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-60nm with 1% trisodium citrate of 2-3ml;
D) collaurum redissolves liquid: 1% bovine serum albumin(BSA), 0.5% degreasing dry powder, 0.05% sodium azide NaN3,1% Tween-20, the 0.01MpH7.0 phosphate buffer, the 0.22U membrane filtration, put 2-8 ℃ standby, the term of validity 15 days;
E) CyMV immune colloid gold solution: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg CyMV monoclonal antibody in the 100ml colloidal gold solution, mix, and the adding bovine serum albumin(BSA) seals, making its final concentration is 0.5%-2%, the centrifugal supernatant that goes, the collaurum redissolution liquid of adding 1/10th volumes obtains CyMV immune colloid gold solution;
F) ORSV immune colloid gold solution: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg ORSV monoclonal antibody in the 100ml colloidal gold solution, mix, and the adding bovine serum albumin(BSA) seals, making its final concentration is 0.5%-2%, the centrifugal supernatant that goes, the collaurum redissolution liquid of adding 1/10th volumes obtains ORSV immune colloid gold solution;
G) with e) the CyMV immune colloid gold solution and the f of step) the ORSV immune colloid gold solution of step mixes and adds a certain amount of collaurum and redissolve liquid, until under electron microscope, observing CyMV and ORSV immune colloid gold solution evenly mixes, above-mentioned mixed solution evenly is layered on 22-50cm for every milliliter
2Glass fibre on, dry back forms CyMV and ORSV immune colloid gold pad;
H) CyMV and ORSV monoclonal anti liquid solution are put on the detection line of nitrocellulose membrane respectively in order, goat-anti rabbit polyclonal antibody point is on the nature controlling line of nitrocellulose membrane;
I) sample pad of respectively absorbent filter being made 1, CyMV and ORSV immune colloid gold pad 2, nitrocellulose filter 3 and thieving paper 4 paste on the PVC offset plate 5 of a band adhesive sticker successively, become test strips; And on test strips, be stained with and have the transparent non-setting adhesive that inserts the highest spacing index line MAX of liquid levels;
J) test strips of drying is packed preservation with foil sealing.
Among Fig. 1 nitrocellulose membrane 3 be marked with CyMV monoclonal anti liquid solution point on the detection line 6 and ORSV monoclonal anti liquid solution point on detection line 7, and goat-anti rabbit polyclonal antibody point is on nature controlling line 8.
Detect the method that orchid virus CyMV and ORSV immunity colloidal gold test paper strip detect orchid virus simultaneously with the present invention, may further comprise the steps:
1, testing sample Collecting and dealing: take by weighing orchid blade (or colored, root) 0.5g and add the 0.01MpH7.4 phosphate buffer in 2ml sample pipe, smash to pieces with glass rod, 8000rpm is centrifugal 5 minutes then, gets supernatant, is testing sample solution.
2, orchid virus CyMV will be detected simultaneously before detecting and the ORSV immunity colloidal gold test paper strip returns to room temperature;
3, the sample area that detects orchid virus CyMV and ORSV immunity colloidal gold test paper strip at the same time splashes into the sample solution of 3 steps 1 preparations, or with the sample solution of this test strips inserting step 1 preparation, liquid level is no more than index line MAX(such as Fig. 2,3);
4, react result of determination after 3-30 minute.
On packing material of the present invention, be provided with template drawing,, the nature controlling line red stripes only arranged among Fig. 2, for there not being the template drawing of virus as Fig. 2, shown in Figure 3; Indicate the red stripes of CyMV monoclonal antibody viewing area, the red stripes of ORSV monoclonal antibody viewing area, the red stripes of nature controlling line in the left hand view of Fig. 3 respectively, show to have CyMV and ORSV virus simultaneously; Indicate the red stripes of ORSV monoclonal antibody viewing area, the red stripes of nature controlling line in the right part of flg of Fig. 3, showing only has ORSV virus.
The nature controlling line of orchid virus CyMV and ORSV immunity colloidal gold test paper strip shows red stripes, the CyMV monoclonal antibody does not have red stripes in the viewing area if detect at the same time, does not have CyMV virus quantity contained in CyMV virus or the sample solution to be lower than in the interpret sample solution and detects orchid virus CyMV and ORSV immunity colloidal gold test paper strip detection lower limit simultaneously.
The nature controlling line of orchid virus CyMV and ORSV immunity colloidal gold test paper strip shows red stripes, the ORSV monoclonal antibody does not have red stripes in the viewing area if detect at the same time, does not have ORSV virus quantity contained in ORSV virus or the sample solution to be lower than in the interpret sample solution and detects orchid virus CyMV and ORSV immunity colloidal gold test paper strip detection lower limit simultaneously.
If red stripes all occurs, then contain two kinds of viruses of CyMV and ORSV in the interpret sample solution, and content has all surpassed the detection lower limit in CyMV monoclonal antibody viewing area and ORSV monoclonal antibody viewing area.
If red stripes or there is red stripes ORSV monoclonal antibody viewing area is only arranged in CyMV monoclonal antibody viewing area, then contain wherein a kind of virus and virus quantity in the interpret sample solution and surpass to detect and roll off the production line.
Claims (1)
1. detect the preparation method of orchid virus CyMV and ORSV immunity colloidal gold test paper strip simultaneously, comprise the steps:
A) CyMV MONOCLONAL ANTIBODIES SPECIFIC FOR: obtain the CyMV virus coat protein gene by the homologous clone method, make up prokaryotic expression carrier, obtain the external source fusion of CyMV-CP by abduction delivering, perhaps that expression and purification is good external source fusion injection mouse, the preparation fused cell, and by screening, the high monoclonal antibody of preparation specificity, purification antibody, purity is not less than 95%;
B) ORSV MONOCLONAL ANTIBODIES SPECIFIC FOR: obtain the ORSV virus coat protein gene by the homologous clone method, make up prokaryotic expression carrier, obtain the external source fusion of ORSV-CP by abduction delivering, perhaps that expression and purification is good external source fusion injection mouse, the preparation fused cell, and by screening, the high monoclonal antibody of preparation specificity, purification antibody, purity is not less than 95%;
C) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-60nm with 1% trisodium citrate of 2-3ml;
D) collaurum redissolves liquid: 1% bovine serum albumin(BSA), 0.5% degreasing dry powder, 0.05% sodium azide NaN3,1% Tween-20, the 0.01MpH7.0 phosphate buffer, the 0.22U membrane filtration, put 2-8 ℃ standby;
E) CyMV immune colloid gold solution: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg CyMV monoclonal antibody in the 100ml colloidal gold solution, mix, and the adding bovine serum albumin(BSA) seals, making its final concentration is 0.5%-2%, the centrifugal supernatant that goes, the collaurum redissolution liquid of adding 1/10th volumes obtains CyMV immune colloid gold solution;
F) ORSV immune colloid gold solution: use 0.1mol/LK
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg ORSV monoclonal antibody in the 100ml colloidal gold solution, mix, and the adding bovine serum albumin(BSA) seals, making its final concentration is 0.5%-2%, the centrifugal supernatant that goes, the collaurum redissolution liquid of adding 1/10th volumes obtains ORSV immune colloid gold solution;
G) with e) the CyMV immune colloid gold solution and the f of step) the ORSV immune colloid gold solution of step mixes and adds a certain amount of collaurum and redissolve liquid, until under electron microscope, observing CyMV and ORSV immune colloid gold solution evenly mixes, above-mentioned mixed solution evenly is layered on 22-50cm for every milliliter
2Glass fibre on, dry back forms CyMV and ORSV immune colloid gold pad;
H) CyMV and ORSV monoclonal anti liquid solution are put on the detection line of nitrocellulose membrane respectively in order, goat-anti rabbit polyclonal antibody point is on the nature controlling line of nitrocellulose membrane;
I) sample pad of respectively absorbent filter being made, CyMV and ORSV immune colloid gold pad, nitrocellulose filter and thieving paper paste on the PVC offset plate of a band adhesive sticker successively, become test strips; And on test strips, be stained with and have the transparent non-setting adhesive that inserts the highest spacing index line MAX of liquid levels;
J) test strips of drying is packed preservation with foil sealing.
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Application publication date: 20110504 |