CN101294962A - Immune colloidal gold reagent for detecting orchid virus series and its preparation - Google Patents
Immune colloidal gold reagent for detecting orchid virus series and its preparation Download PDFInfo
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- CN101294962A CN101294962A CNA2007100088900A CN200710008890A CN101294962A CN 101294962 A CN101294962 A CN 101294962A CN A2007100088900 A CNA2007100088900 A CN A2007100088900A CN 200710008890 A CN200710008890 A CN 200710008890A CN 101294962 A CN101294962 A CN 101294962A
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Abstract
The invention discloses an immune colloidal gold test strip used for detecting cymbidium virus series and preparing method thereof, comprising an immune colloidal gold test strip used for detecting cymbidium CyMV virus and ORSV virus and preparing method thereof; the immune colloidal gold test strip used for detecting cymbidium virus series has the advantages of accurate result, fast determination, simple and convenient operation and low cost, etc., does not need washing process and standard contrast, can detect the samples in batches or singly, and is easy for promotion and use; the operator does not need professional training and can complete the operation according to the specification.
Description
Technical field
The present invention relates to a kind of with the immune colloid gold method detect the orchid mosaic virus (Cymbium mosaic virus, CyMV) and odontoglossum ring spot virus (Odontoglossum ringspotvirus, reagent ORSV) and preparation method thereof.
Background technology
Orchid is world-renowned flowers.There are ten thousand of orchid initial species 3-3.5 in the whole world, 800 genus, ten thousand kinds of 6-8, many orchids all have higher gardening and ornamental value, have the title of " green stock ", China mainland begins to earn foreign exchange for export than the commerial growing orchid in some provinces and cities in recent years.Multiple orchid is also planted in China Taiwan, and its technology and scale all are in the leading level in the world.
But in process of production, orchid usually is subjected to the harm of virosis, can cause symptoms such as orchid plant tikka, necrosis and flower variable color, deformity, has a strong impact on its quality, and its commodity value is reduced greatly, has a strong impact on the development of orchid industry.This situation has played the attention of countries in the world already.External many scholars have carried out big quantity research to the orchid virus disease, reported from orchid so far and be separated at least 25 kinds of viruses, that wherein distribution is the widest, harm is the heaviest is cymbidium mosaic virus (Cymbium mosaicvirus, CyMV) and odontoglossum ring spot virus (Odontoglossum ringspot virus, ORSV).As far back as 1997, China's animal and plant quarantine department was just classified the orchid virus disease as the harmful organism of being potentially dangerous property.But orchid virus does not have the medicament of radical cure to come out so far as yet, so the orchid virus disease is also referred to as " orchid cancer ".
Long-term production facts have proved that the production of orchid virus-elimination seedlings is that virus disease endangers one of serious effective means in the solution orchid production.In recent years, along with the development of tissue culture technique, the production unit of China's orchid detoxic seedling increases gradually, and the virus-elimination seedlings cultivated area constantly enlarges, and part orchid product also exports to foreign countries.But most of seedling production units wherein do not have viral testing conditions so that market on sell seedling quality uneven.Because importer is very strict to the quarantine request of orchid, and China's Check and Examination of Port quarantine departments lacks effective detection method, return of goods phenomenon often occurs, bring about great losses for enterprise and country.Therefore set up the program of fast detecting virus, find out the morbidity strain, artificially destroy or isolate, become one of the effective measure that virosis is further propagated, spread that prevents.Thus, quick, accurate, special, sensitive viral detection means is to improve the task of top priority and the powerful guarantee of flowers quality.
Detection method to orchid virus mainly comprises Biology identification, enzyme linked immunosorbent assay, RT-PCR method at present.
1, Biology identification is a kind of very accurate and reliable method, but also has following shortcoming: (1) different virus and different hosts, need to adopt some special inoculation measures ability successes, and the technical requirement difficulty is higher; (2) the whole qualification process cycle oversize, such as the time of inoculation back symptom performance longer, generally take more than 3 days, some host's symptom performance needs 20 to 24 days; (3) performance of inoculation back symptom also is subjected to the external environment factor affecting; (4) need to operate through the technician of professional training, operating personnel will have abundant correlation experience, also need understand the various disturbing factors of biological assay, understand and judge the correctness of biological symptom, could obtain reliable analysis result; (5) though do not need valuable instrument and equipment, need relatively independent and clean plant culture space, infect between the different virus avoiding.
2, or enzyme linked immunosorbent assay is to serve as to detect principle with indirect ELISA double antibodies sandwich ELISA, with the double antibodies sandwich method is example, it is with tobacco mosaic virus (TMV) antibody sandwich ELISA Plate, during detection detected sample is added ELISA Plate, the OD value is surveyed by colour developing at last with adding ELIAS secondary antibody again in the reaction back.The shortcoming that exists is: (1) needs special instrument and equipment such as microplate reader to be used, and is unfavorable for promoting and using in grass-roots unit; (2) the detecting operation personnel need pass through professional training; (3) operating process is relatively complicated, and it is long to detect required time, and whole process needs 4h-8h; (4) the required expense of detection is higher, can not realize that single sample detects.
3, the RT-PCR method is based on round pcr, directly to the nucleotide sequence of virus detect the .RT-PCR method with this viral capsid viral gene as the primer template, synthetic one primer of forming by 20 base-pairs.Then viral nucleotide is increased, come whether to exist in the judgement sample this virus whether to produce this amplified production in the reaction system.So this method sensitivity all has significant improvement than preceding method, it is few that sensitivity can reach the required sample size of 10fg., be suitable for detecting the low content sample, but simultaneously also there is following shortcoming in the RT-PCR method: instrument and equipment such as PCR instrument that (1) needs are special are used, and are unfavorable for promoting and using in grass-roots unit; (2) requirement of experiment is relatively strict, and the detecting operation personnel need pass through professional training; (3) operating process is relatively complicated, and whole process needs 3h-5h; (4) the required expense of detection is higher, can not realize that single sample detects.
Summary of the invention
The objective of the invention is, a kind of immune colloid gold reagent that is applicable to orchid virus CyMV and ORSV and preparation method thereof is provided, with solve that prior art exists long as detection time, can not on-the-spot detection, shortcoming such as testing cost height.
The preparation method of detection orchid virus series immune colloid gold reagent of the present invention comprises the preparation method of the immunity colloidal gold test paper strip that detects CyMV virus and detects the preparation method of the immunity colloidal gold test paper strip of ORSV virus.
1, detect the preparation method of the immunity colloidal gold test paper strip of orchid CyMV virus, may further comprise the steps:
(1) CyMV polyclonal antibody: get the CyMV virus injection rabbit of purifying, serum is got in blood sampling, the purification IgG antibody, and purity is not less than 95%;
(2) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-40nm with 1% trisodium citrate of 2-3ml;
(3) CyMV immune colloid gold solution: use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg CyMV polyclonal antibody in the 100ml colloidal gold solution, mix, get CyMV immune colloid gold solution;
(4) described CyMV immune colloid gold solution is put on the glass fibre; Form CyMV immune colloid gold pad;
(5), put respectively on the diverse location of nitrocellulose membrane with CyMV polyclonal antibody solution and goat-anti rabbit polyclonal antibody; Drip the CyMV polyclonal antibody arranged be called CyMV polyclonal antibody viewing area, dripping has the position of goat-anti rabbit polyclonal antibody to be called the nature controlling line viewing area; Described goat-anti rabbit polyclonal antibody can be bought in the shop.
(6) respectively sample pad, CyMV immune colloid gold pad, nitrocellulose filter and thieving paper are pasted on the offset plate of an adhesive sticker successively, be stained with adhesive sticker and index line again at two ends, cut into test strips then;
(7) with described test strips vacuum drying, the temperature in the vacuum pump is that 15-25 ℃, drying time are 2-5 hour; Atmospheric pressure in the vacuum pump is not higher than 0.1mba;
(8) test strips of drying is packed with foil sealing get final product.
2, detect the preparation method of the immunity colloidal gold test paper strip of orchid ORSV virus, may further comprise the steps:
(1) ORSV polyclonal antibody: get the ORSV virus injection rabbit of purifying, serum is got in blood sampling, the purification IgG antibody, and purity is not less than 95%;
(2) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-40nm with 1% trisodium citrate of 2-3ml;
(3) ORSV immune colloid gold solution: use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, press adding 0.5mg-2mg ORSV polyclonal antibody in the 100ml colloidal gold solution, mix, get ORSV immune colloid gold solution;
(4) above-mentioned ORSV immune colloid gold solution is put on the glass fibre; Form ORSV immune colloid gold pad;
(5), put respectively on the diverse location of nitrocellulose membrane with ORSV polyclonal antibody solution and goat-anti rabbit polyclonal antibody; Drip the ORSV polyclonal antibody arranged be called ORSV polyclonal antibody viewing area, drip positions that have the many grams of goat-anti rabbit to fall antibody and be called the nature controlling line viewing area; The many grams of described goat-anti rabbit fall antibody and can buy in the shop.
(6) respectively sample pad, ORSV immune colloid gold pad, nitrocellulose filter and thieving paper are pasted on the offset plate of an adhesive sticker successively, be stained with adhesive sticker and index line again at two ends, cut into test strips then;
(7) with described test strips vacuum drying, the temperature in the vacuum pump is 15-25 ℃; Be 2-5 hour drying time; Atmospheric pressure in the vacuum pump is not higher than 0.1mba;
(8) test strips of drying is packed with foil sealing get final product.
Good effect of the present invention is:
(1) low price: the production flow process is simple, production cost is low, the expense that detects only needs the 1/5-1/10 of RT-PCR, (RT-PCR detects a sample usually needs 200 yuan-300 yuan also to want considerably cheaper than enzyme marking reagent box, the common sample detection of enzyme marking reagent box also needs 50 yuan-80 yuan, and the immune colloid gold reagent box of the present invention's preparation only needs 15 yuan-50 yuan usually);
(2) detection speed is fast: overall process only needs 30 minutes, can realize that the oneself detects;
(3) can on-the-spotly detect;
(4) high-quality: good, highly sensitive, the good reproducibility of the kit specificity that this method is prepared from;
(5) easy and simple to handle: the kit that this method is prepared from is cue mark with the collaurum, fast qualitative, the result is accurately, fast, and is easy and simple to handle, need not flushing process and standard control, can be in batches or single sample in time detect;
(6) be easy to promote the use of: operating personnel need not professional training, and by specification gets final product complete operation.
(7) can carry out the detection of single virus, also can carry out the detection of a plurality of viruses simultaneously, shorten detection time and step, also save material.
Specific embodiments is illustrated below in conjunction with embodiment in order fully to disclose detection orchid virus series immunity colloidal gold test paper strip of the present invention and preparation method thereof.
Embodiment 1: utilize the CyMV immunity colloidal gold test paper strip to detect the method for orchid CyMV virus, may further comprise the steps:
1) testing sample Collecting and dealing: take by weighing sample blade 0.5 gram and put into mortar, sample buffer is poured into ground in the mortar, extracting sample solution 10000rpm was centrifugal 5 minutes after grinding finished, and got supernatant, i.e. the detected sample solution for standby.
2) detect:
Before a, the test CyMV immunity colloidal gold test paper strip is returned to room temperature;
B, drip 3-5 in the polyclonal antibody viewing area of CyMV immune colloid gold reagent bar and drip detected sample solution;
C, reaction result of determination after 5 minutes～30 minutes.
3) interpretation as a result:
If red stripes does not appear in the CyMV polyclonal antibody viewing area of CyMV immune colloid gold reagent bar, contain the detection lower limit of the amount of virus in orchid CyMV virus that then will not detect in the sample or the sample less than the CyMV immunity colloidal gold test paper strip; If red stripes appears in the CyMV polyclonal antibody viewing area of CyMV immune colloid gold reagent bar, then orchid CyMV virus that existence will detect in the sample and viral level surpass the detection lower limit of CyMV immunity colloidal gold test paper strip.
Embodiment 2: utilize the ORSV immunity colloidal gold test paper strip to detect the method for orchid ORSV virus
Utilize the ORSV immunity colloidal gold test paper strip to detect the method for orchid ORSV virus: change the CyMV immunity colloidal gold test paper strip among the embodiment 1 into the ORSV immunity colloidal gold test paper strip, all the other detection methods are the same with the detection method of embodiment 1.
Claims (4)
1, detect the preparation method of the immunity colloidal gold test paper strip of orchid CyMV virus, may further comprise the steps:
(1) CyMV polyclonal antibody: get the CyMV virus injection rabbit of purifying, serum is got in blood sampling, the purification IgG antibody, and purity is not less than 95%;
(2) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-40nm with 1% trisodium citrate of 2-3ml;
(3) CyMV immune colloid gold solution: use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, colloidal gold solution is pressed adding 0.5mg-2mg CyMV polyclonal antibody in the 100ml colloidal gold solution, mix, get CyMV immune colloid gold solution;
(4) described CyMV immune colloid gold solution is put on the glass fibre; Form CyMV immune colloid gold pad;
(5), put respectively on the diverse location of nitrocellulose membrane with CyMV polyclonal antibody solution and goat-anti rabbit polyclonal antibody;
(6) respectively sample pad, CyMV immune colloid gold pad, nitrocellulose filter and thieving paper are pasted on the offset plate of an adhesive sticker successively, be stained with adhesive sticker and index line again at two ends, cut into test strips then;
(7) with described test strips vacuum drying, the temperature in the vacuum pump is that 15-25 ℃, drying time are 2-5 hour; Atmospheric pressure in the vacuum pump is not higher than 0.1mba;
(8) test strips of drying is packed with foil sealing get final product.
2, utilize the immunity colloidal gold test paper strip of detection orchid CyMV virus of the method preparation of claim 1.
3, detect the preparation method of the immunity colloidal gold test paper strip of orchid ORSV virus, may further comprise the steps:
(1) ORSV polyclonal antibody: get the ORSV virus injection rabbit of purifying, serum is got in blood sampling, the purification IgG antibody, and purity is not less than 95%;
(2) collaurum: the colloidal gold solution that 100ml 0.01% chlorauride is reduced into 15nm-40nm with 1% trisodium citrate of 2-3ml;
(3) ORSV immune colloid gold solution: use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7-8.5, press adding 0.5mg-2mg ORSV polyclonal antibody in the 100ml colloidal gold solution, mix, get ORSV immune colloid gold solution;
(4) above-mentioned ORSV immune colloid gold solution is put on the glass fibre; Form ORSV immune colloid gold pad;
(5), put respectively on the diverse location of nitrocellulose membrane with ORSV polyclonal antibody solution and goat-anti rabbit polyclonal antibody;
(5), put respectively and be provided with ORSV polyclonal antibody viewing area and nature controlling line viewing area on the nitrocellulose membrane with ORSV polyclonal antibody solution and goat-anti rabbit polyclonal antibody;
(6) respectively sample pad, ORSV immune colloid gold pad, nitrocellulose filter and thieving paper are pasted on the offset plate of an adhesive sticker successively, be stained with adhesive sticker and index line again at two ends, cut into test strips then;
(7) with described test strips vacuum drying, the temperature in the vacuum pump is 15-25 ℃; Be 2-5 hour drying time; Atmospheric pressure in the vacuum pump is not higher than 0.1mba;
(8) test strips of drying is packed with foil sealing get final product.
4, utilize the immune colloid gold test paper of detection orchid ORSV virus of the method preparation of claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100088900A CN101294962A (en) | 2007-04-26 | 2007-04-26 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100088900A CN101294962A (en) | 2007-04-26 | 2007-04-26 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043056A (en) * | 2010-11-10 | 2011-05-04 | 福建省亚热带植物研究所 | Preparation method for immune colloidal gold test strip for simultaneously detecting orchid viruses CyMV and ORSV |
| CN102253205A (en) * | 2011-06-16 | 2011-11-23 | 昆明倍尔遵生科技有限公司 | Colloidal gold test paper strip for detecting respiratory syncytial virus antibody and preparation method thereof |
| CN103412121A (en) * | 2013-07-20 | 2013-11-27 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
-
2007
- 2007-04-26 CN CNA2007100088900A patent/CN101294962A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043056A (en) * | 2010-11-10 | 2011-05-04 | 福建省亚热带植物研究所 | Preparation method for immune colloidal gold test strip for simultaneously detecting orchid viruses CyMV and ORSV |
| CN102253205A (en) * | 2011-06-16 | 2011-11-23 | 昆明倍尔遵生科技有限公司 | Colloidal gold test paper strip for detecting respiratory syncytial virus antibody and preparation method thereof |
| CN103412121A (en) * | 2013-07-20 | 2013-11-27 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
| CN103412121B (en) * | 2013-07-20 | 2015-02-04 | 福建农林大学 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
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Open date: 20081029 |