CN103412121A - Colloidal gold immune test strip for rapid detection of Papaya ringspot virus - Google Patents
Colloidal gold immune test strip for rapid detection of Papaya ringspot virus Download PDFInfo
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Abstract
The invention relates to preparation and application of an immune colloidal gold test strip for rapid detection of Papaya ringspot virus (PRSV). The preparation method includes: taking PRSV-Vb bred on summer squashes as a material, adopting purified PRSV as an antigen for preparation of a polyclonal antibody, and labelling the polyclonal antibody by colloidal gold to form a gold-labeled antibody, coating the detection line and quality control line of a nitrocellulose membrane respectively with the polyclonal antibody and goat anti-rabbit IgG, coating a combination pad prepared from a glass cellulose membrane with the gold-labeled antibody, then sticking the nitrocellulose membrane coated by the polyclonal antibody and the goat anti-rabbit IgG on a PVC base plate, finally sticking the gold-labeled combination pad and a sample pad prepared from glass cellulose at the detection line end in order, and sticking absorbent paper at the quality control line end, thus obtaining the PRSV colloidal gold immune test strip. The colloidal gold immune test strip provided in the invention has the advantages of simple operation, fast detection speed, no need of special equipment, accurate and reliable detection result, and low cost, and can be used for early diagnosis of field diseased plants.
Description
Technical field
The invention belongs to plant disease etiological diagnosis technical field, relate to the fast diagnosis method of a kind of plant virus, particularly relate to a kind of preparation and application that detect the PRSV immunity colloidal gold test paper strip.
Background technology
PRSV (Papaya ringspot virus, PRSV) is the important member of Potyvirus, is one of important virus caused papaya virosis.The forties in 20th century U.S.'s reported first this virus.China has introduced papaya in early 1950s and has carried out plant development, and nineteen fifty-nine has just been found on papaya should virus, and after 1962, PRSV is widely current in China.In a single day the papaya plant is subject to infecting of PRSV, and mortality ratio can, up to 90%, make papaya production be subject to destructive strike.Thousands of mu of papaya of Hainan Province's plantation in 2000 are because of the almost total crop failure of infecting of PRSV.Due to seriously causing harm of PRSV, a plurality of regional papaya production of China is forced to the planting patterns of taking the spring planting autumn to cut, when being spring by under papaya seedling kind, and tree is cut down in autumn the time, make perennial papaya people for becoming annual planting, not only give to produce and brought very large trouble, also caused serious economic loss simultaneously.The early diagnosis in field is the key link of the effective prevention and control of the viroses of plant.At present, the method for the Resisting Ringspot Virus of Papaya etiological diagnosis mainly contains traditional biological method, enzyme-linked immunosorbent assay (ELISA) and molecular biology method.Although traditional biological method identifying virus is simple to operate, without special instruments and equipment, but have the following disadvantages: (1) needs to adopt a series of differential host to identify, and the layman often is faced with the problem that the symptom occurred is difficult to accurate judgement.(2) in reality is identified, often multiple viral Combined Infection host, produce greatly symptom and change, and is difficult for judgement; (3) evaluation speed is slow, and the cycle is long.After inoculation, the differential host produces the required time of symptom generally more than 3 days, and what have even reached more than 20 days; (4) qualification result is subject to the impact of season and surrounding enviroment, and needs a relatively independent space, avoids viral cross infection; (5) some virus there is no suitable differential host, can't adopt the traditional biological method to identify.Though detecting virus, enzyme-linked immunosorbent assay and molecular biology method have advantages of that detection specificity is strong, highly sensitive, detection time is short than the traditional biological method, but at least also have the following disadvantages: (1) these two kinds of methods all need special instrument and equipment, and operating personnel generally will pass through special training, make these the two kinds viral methods of detection in laboratory, to complete, be unfavorable for promoting and using in grass-roots unit; (2) with the traditional biological method, compare, though very large shortening is arranged detection time, required time is at least also wanted a few hours, still is not suitable for the fast detecting at scene, field; (3) detect required cost higher, particularly molecular biology for detection, need to use expensive reagent.The colloid gold immune test paper bar adopts colloidal gold-labeled method to develop, and collaurum is gold chloride (HAuCl
4) the hydrosol, gold chloride aggregates into the gold grain of specific size under the effect of reductive agent, and because electrostatic interaction becomes a kind of stable colloidal state.Colloidal gold-labeled method is exactly by regulating the addition of reductive agent, prepare the colloid gold particle of different sizes and color, these colloid gold particles have very strong adsorbability to albumen, and the formation naked eyes of can being combined with the immunoglobulin (Ig) non-covalent bond can be seen red or pink color.The advantage of colloid gold immune test paper bar detection method is without by laboratory equipment and professional and technical personnel, the water that only need to take a morsel drops on the sample pad on test strips, just can even in a few minutes, obtain testing result at tens minutes, the characteristics such as have safe and simple, no radioactivity pollute, highly sensitive, result is accurate, cost is low, be applicable to the Site Detection to plant virus.
Summary of the invention
The present invention aims to provide a kind of preparation method of colloidal gold immuno-chromatography test paper strip of fast detecting PRSV, solves current field field quick detection PRSV and still lacks the effective means problem.That test strips prepared by the present invention has is simple to operate, detect fast, result accurately and reliably, with low cost, without the instrument and equipment by special, be applicable to the characteristics such as field Site Detection, can be used for the early diagnosis of field diseased plant, for the control of Resisting Ringspot Virus of Papaya provides effective early warning, improve the efficiency of preventing and treating of Resisting Ringspot Virus of Papaya, reduce by its economic loss caused.
A kind of detection PRSV immunity colloidal gold test paper strip, is characterized in that the preparation method comprises the steps:
1) purification of PRSV: PRSV-Vb strain frictional inoculation is infected on host's cucurbita pepo in system, and the cucurbita pepo blade that classical symptom occurs of take is purified as the material of purification PRSV, obtains the PRSV of purification;
2) preparation of polyclonal antibody: the PRSV that the step 1) of take is purified is antigen, and Freunds adjuvant is emulsifying agent, immune new zealand white rabbit according to a conventional method, the polyclonal antibody of the anti-PRSV of preparation;
3) polyclonal antibody purifying: to step 2) prepared anti-PRSV polyclonal antibody carries out purifying;
4) preparation of colloidal gold solution: the tetra chlorauric acid solution in 100 mL 0.01% w/v prepares colloidal gold solution by the ratio of the 1% w/v sodium citrate solution reduction of 1.5 mL – 3 mL, solution stirs and boils 15 min, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions;
5) preparation of PRSV gold labeling antibody: get colloidal gold solution prepared by 100 mL step 4), use 0.1M K
2CO
3Regulate colloidal gold solution pH value to 7.2 – 8.0, then add while stirring the antibody of 1.5 mg – 2 mg step 3) purifying, stir and evenly mix, add 5% w/v bovine serum albumin to seal, addition is 1% w/v for making the bovine serum albumin final concentration; Solution after sealing in 250 *
gCentrifugal 20 min – 30 min, discard the formed precipitation of gold grain of gathering, supernatant in 14000 *
gCentrifugal 40 min – 60 min, abandoning supernatant, precipitation adds buffer suspension liquid to 100 mL and suspends; Buffer suspension liquid is: contain 1% w/vBSA and 0.02% w/v NaN
30.01 M pH 7.2-8.0 phosphate buffer, the filtrate after 0.22 μ m filtering with microporous membrane is preserved under 4 ℃; Solution after suspension repeats high speed centrifugation once, abandoning supernatant, and precipitation is suspended to 10 mL with above-mentioned same buffer suspension liquid again, and with 0.22 μ m filtering with microporous membrane, filtrate is PRSV gold labeling antibody solution, preserves under 4 ℃ of conditions;
6) preparation of PRSV gold labeling antibody pad: 4 mm that will cut * 6 mm glass fibre element films are immersed in 30 min in PRSV gold labeling antibody pad sealing damping fluid, 37 ℃ of dry 30-60 min in advance; The golden labeling antibody solution 10 – 20 μ L mixing liquids of getting after dilution are sprayed on the glass fibre element film that above-mentioned pre-service is good, and 37 ℃ of drying 30 min – 2 h, make PRSV gold labeling antibody pad; The preparation of wherein said PRSV gold labeling antibody pad sealing damping fluid: 0.2% w/v BSA, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions; The preparation of the golden labeling antibody solution after described dilution: get PRSV gold labeling antibody solution 1 mL prepared by step 5), add PRSV gold labeling antibody dilution buffer liquid 1 mL – 3 mL and mix; Described PRSV gold labeling antibody dilution buffer liquid be prepared as 1% w/v BSA, 2.5% w/v sucrose, 0.01 M pH 7.2 – 8.0 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collection filtrate is preserved under 4 ℃ of conditions;
7) making of detection line and nature controlling line: it is 1.6 – 2.0 mg/mL that the antibody after the step 3) purifying is diluted to concentration with 0.01 M pH 8.0 phosphate buffers, it is 0.5 – 0.6 mg/mL that goat anti-rabbit igg is diluted to concentration with 0.01 M pH 8.0 phosphate buffers, then the PRSV antibody after diluting and goat anti-rabbit igg are put respectively on nitrocellulose filter detection line and nature controlling line with 2.5 μ L/cm, two lines, 5 mm of being separated by, 37 ℃ of drying 30 – 60 min; Dried nitrocellulose filter is immersed in cellulose nitrate membrane closure damping fluid, hatches 1 – 2 h for 37 ℃, after taking-up, wash 3 times with the phosphate buffer of 0.01M pH 7.0, room temperature is dried; The preparation of cellulose nitrate membrane closure damping fluid: 0.5%W/V PVP, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions;
8) nitrocellulose filter of first step 7) being processed is pasted on the centre position of PVC base plate, then toward the end of PVC, paste successively the sample pad that PRSV gold labeling antibody pad and glass fibre element film are made, the other end pastes pastes thieving paper as the Quality Control line end;
9) after the test strips drying prepared by step 8), be the immunity colloidal gold test paper strip of PRSV, use foil sealing, 4 ℃ of preservations.
The purification specific practice of described PRSV is as follows:
PRSV – Vb strain frictional inoculation is infected on host's cucurbita pepo in system, take cucurbita pepo blade that classical symptom the occurs material as purification PRSV; Get the sick leaf of 100-500 g in mortar, add liquid nitrogen to not having sick leaf, rapidly with grinding rod by sick leaf grind into powder, by powder transfer, to beaker, be the phosphate buffer A that 1:2-3 adds 4 ℃ of lower precoolings by mass volume ratio; Described phosphate buffer A concentration is 0.5M, and pH7.2 separately contains 0.1 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3%w/v Na
2SO
3, 0.5% w/v tritonX-100; Under 4 ℃, stir 1h and must be suspended damping fluid, by being suspended damping fluid: mixeding liquid volume is than for 5:3, adding CCl
4With CHCl
3The equal-volume mixed liquor, stir 30 min under 4 ℃, 3000 *
gCentrifugal 15 min, get upper solution, upper solution through 6000 *
gCentrifugal 15 min, get in supernatant C and add PEG6000 and NaCl to be respectively 6% w/v and 3% w/v to final concentration, after stirring and dissolving under 4 ℃ standing 2-10 h, 6000 *
gCentrifugal 15-30 min, go supernatant to stay precipitation, adds the phosphate buffer B of the 0.1M pH7.2 of supernatant C 1/5 volume; Described phosphate buffer B concentration is 0.1M, and pH7.2 separately contains 0.01 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3% Na
2SO
3, 0.5% tritonX-100; 4 ℃ of lower magnetic forces stir 4-8 h, 10000 *
gCentrifugal 15 min, get supernatant, in supernatant, adds PEG6000 and NaCl to be respectively 6% w/v and 3% w/v to final concentration, after stirring and dissolving under 4 ℃ standing 2-6 h, 6000 *
gCentrifugal 15 min, go supernatant to stay precipitation, adds supernatant C 1/20 V
0Phosphate buffer B, stir 1-2 h under 4 ℃, 8000 *
gCentrifugal 15 min, get supernatant, and supernatant carefully is added on the sucrose pad into 20%w/v, 110000 *
gCentrifugal 1.5-2 h, go supernatant to get precipitation, and precipitation is viral crude extract after adding the phosphate buffer of 2-4 mL0.1M pH7.2 to dissolve; In centrifuge tube, add successively each 3 mL of 30% sucrose solution that contain 0.7M, 0.5M, 0.3M and 0M cesium sulfate, standing 8-12 h in 4 ℃ of refrigerators, make the sucrose cesium sulfate solution form continuous gradient, viral crude extract be added on continuous gradient solution, immediately 4 ℃ 160000 *
gLower centrifugal 3 h, take out centrifuge tube, with electric torch, irradiates the visible glassy yellow virus of centrifuge tube band in darkroom, carefully draws the solution at viral band position, with after 1-3 mL phosphate buffer B dilution, 4 ℃ 100000 *
gLower centrifugal 1-2 h, precipitation is the PRSV of purification.
Described polyclonal antibody purifying is specially: the anti-PRSV polyclonal antibody that will prepare adopts Protein A affinity column to carry out purifying.Can select Thermo company
Protein A Columns, and carry out purifying by its instructions purification step;
Described test strips is characterized in that usage is:
1) will treat that measuring plants gets the phosphate buffer 3-6 mL that 0.5g adds 0.01 M pH 7.0, fully grind, then centrifuging and taking supernatant, i.e. testing sample solution;
2) after the PRSV immunity colloidal gold test paper strip is recovered to room temperature, the sample pad of test strips is immersed in the testing sample solution of preparation, or in the sample pad place, drip testing sample solution prepared by about 200 μ L, react result of determination after 5 – 30 min minutes;
Result is judged: if redness all appears in the detection line of test strips and nature controlling line place, illustrate in this sample that the content that contains PRSV and virus surpasses the detection lower limit of immunity colloidal gold test paper strip; Red stripes does not appear in the detection line place if the nature controlling line place manifests red stripes, and the content that do not contain PRSV or PRSV in this sample detection lower limit lower than immunity colloidal gold test paper strip is described; If red stripes does not all appear in detection line and nature controlling line place, illustrate that this test strips lost efficacy, measured result is unavailable.
Notable feature of the present invention is: can in 5-20 min, carry out quick diagnosis to the PRSV in sample by test strips prepared by technology of the present invention, this test strips low production cost, easy and simple to handle, detection speed is fast, result accurately and reliably, can the field Site Detection, do not need special instrument and equipment, operating personnel not to need through special training, be easy to promote the use of, can carry out early diagnosis to the field diseased plant, reduce poison source, field quantity, reduce the economic loss that causes harm and cause because of PRSV.
The accompanying drawing explanation
Fig. 1 is schematic diagram of the present invention
1: the sample pad 2:PRSV gold labeling antibody pad 3 that glass fibre element film is made: nitrocellulose filter 4:PRSV polyclonal antibody point is on the detection line of nitrocellulose filter 5: goat anti-rabbit igg point is 6:PVC base plate 7 on the nitrocellulose filter nature controlling line: thieving paper
Fig. 2 testing result schematic diagram
Be followed successively by from left to right: the testing result positive, detection line (T) and two lines of nature controlling line (C) all develop the color; The testing result feminine gender, the colour developing of nature controlling line (C) line, and detection line (T) does not develop the color; Test strips lost efficacy, and detection line (T) and nature controlling line (C) all do not develop the color.
Embodiment
In order to make content of the present invention more be convenient to understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention is not limited only to this.
Embodiment 1
A kind of detection PRSV immunity colloidal gold test paper strip, is characterized in that the preparation method comprises the steps:
1) PRSV – Vb strain frictional inoculation is infected on host's cucurbita pepo in system, take cucurbita pepo blade that classical symptom the occurs material as purification PRSV; Get the sick leaf of 200 g in mortar, add liquid nitrogen to not having sick leaf, rapidly with grinding rod by sick leaf grind into powder, powder transfer, to beaker, is added to the phosphate buffer A of 4 ℃ of lower precoolings of 500 mL; Described phosphate buffer A concentration is 0.5M, and pH7.2 separately contains 0.1 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3%w/v Na
2SO
3, 0.5% w/v tritonX-100; Under 4 ℃, stir 1h and must be suspended damping fluid, add 300 mL CCl
4With CHCl
3The equal-volume mixed liquor, stir 30 min under 4 ℃, 3000 *
gCentrifugal 15 min, get upper solution, upper solution through 6000 *
gCentrifugal 15 min, get in supernatant 400 mL and add the PEG6000 of 24 g and the NaCl of 12 g, after stirring and dissolving under 4 ℃ standing 8 h, 6000 *
gCentrifugal 15 min, go supernatant to stay precipitation, in supernatant, adds the phosphate buffer B of the 0.1M pH7.2 of 80 mL; Described phosphate buffer B concentration is 0.1M, and pH7.2 separately contains 0.01 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3% Na
2SO
3, 0.5% tritonX-100; 4 ℃ of lower magnetic forces stir 4 h, 10000 *
gCentrifugal 15 min, get supernatant, in supernatant, adds the PEG6000 of 24 g and the NaCl of 12 g, after stirring and dissolving under 4 ℃ standing 2 h, 6000 *
gCentrifugal 15 min, go supernatant to stay precipitation, adds the phosphate buffer B of supernatant 20 mL, stirs 1 h under 4 ℃, 8000 *
gCentrifugal 15 min, get supernatant, and supernatant carefully is added on the sucrose pad into 20%w/v, 110000 *
gCentrifugal 1.5 h, go supernatant to get precipitation, and precipitation is viral crude extract after adding the phosphate buffer of 3 mL0.1M pH7.2 to dissolve; In centrifuge tube, add successively each 3 mL of 30% sucrose solution that contain 0.7M, 0.5M, 0.3M and 0M cesium sulfate, standing 12 h in 4 ℃ of refrigerators, make the sucrose cesium sulfate solution form continuous gradient, viral crude extract be added on continuous gradient solution, immediately 4 ℃ 160000 *
gLower centrifugal 3 h, take out centrifuge tube, with electric torch, irradiates the visible glassy yellow virus of centrifuge tube band in darkroom, carefully draws the solution at viral band position, with after 2 mL phosphate buffer B dilutions, 4 ℃ 100000 *
gLower centrifugal 1.5 h, precipitation is the PRSV of purification.
2) preparation of polyclonal antibody: it is 2 mg/ mL that the PRSV that step 1) is purified is diluted to concentration with the phosphate buffer of 0.1M pH7.2, get the PRSV after 1 mL dilutes, add 1 mL complete Freund's adjuvant, fully emulsified rear intramuscular injection new zealand white rabbit, 10 d after injection for the first time, 17 d, 24 d and 31 d inject respectively PRSV after 1 mL dilution and the emulsion of 1 mL incomplete Freund's adjuvant, after last injection, 10 d get rabbit blood, blood is placed in to 4 ℃ of refrigerators, after serum is separated out, suck the polyclonal antibody that serum is preparation,
3) anti-PRSV polyclonal antibody polyclonal antibody purifying: by step 2) prepared is by Thermo company
Protein A Columns instructions purification step carries out purifying;
4) preparation of colloidal gold solution: the tetra chlorauric acid solution in 100 mL 0.01% w/v prepares colloidal gold solution by the ratio of the 1% w/v sodium citrate solution reduction of 2 mL, solution stirs and boils 15 min, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions;
5) preparation of PRSV gold labeling antibody: get colloidal gold solution prepared by 100 mL step 4), use 0.1M K
2CO
3Regulate colloidal gold solution pH value to 7.6, then add while stirring the antibody of 1.8 mg step 3) purifying, stir and evenly mix, add 5% w/v bovine serum albumin to seal, addition is 1% w/v for making the bovine serum albumin final concentration; Solution after sealing in 250 *
gCentrifugal 20 min, discard the formed precipitation of gold grain of gathering, supernatant in 14000 *
gCentrifugal 50 min, abandoning supernatant, precipitation adds buffer suspension liquid to 100 mL and suspends; Buffer suspension liquid is: contain 1% w/vBSA and 0.02% w/v NaN
30.01 M pH 7.2 phosphate buffers, the filtrate after 0.22 μ m filtering with microporous membrane is preserved under 4 ℃; Solution after suspension repeats high speed centrifugation once, abandoning supernatant, and precipitation is suspended to 10 mL with above-mentioned same buffer suspension liquid again, and with 0.22 μ m filtering with microporous membrane, filtrate is PRSV gold labeling antibody solution, preserves under 4 ℃ of conditions;
6) preparation of PRSV gold labeling antibody pad: 4 mm that will cut * 6 mm glass fibre element films are immersed in 30 min in PRSV gold labeling antibody pad sealing damping fluid, 37 ℃ of drying 30 min in advance; The golden labeling antibody solution 15 μ L mixing liquids of getting after dilution are sprayed on the glass fibre element film that above-mentioned pre-service is good, and 37 ℃ of dry 1h, make PRSV gold labeling antibody pad; The preparation of wherein said PRSV gold labeling antibody pad sealing damping fluid: 0.2% w/v BSA, 0.5%V/V Tween – 20,0.01 M pH 7.6 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions; The preparation of the golden labeling antibody solution after described dilution: get PRSV gold labeling antibody solution 1 mL prepared by step 5), add PRSV gold labeling antibody dilution buffer liquid 3 mL and mix; Described PRSV gold labeling antibody dilution buffer liquid be prepared as 1% w/v BSA, 2.5% w/v sucrose, 0.01 M pH 7.6 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collection filtrate is preserved under 4 ℃ of conditions;
7) making of detection line and nature controlling line: it is 1.6 mg/mL that the antibody after the step 3) purifying is diluted to concentration with 0.01 M pH 8.0 phosphate buffers, it is 0.5 mg/mL that goat anti-rabbit igg is diluted to concentration with 0.01 M pH 8.0 phosphate buffers, then the PRSV antibody after diluting and goat anti-rabbit igg are put respectively on nitrocellulose filter detection line and nature controlling line with 2.5 μ L/cm, two lines, 5 mm of being separated by, 37 ℃ of drying 60 min; Dried nitrocellulose filter is immersed in cellulose nitrate membrane closure damping fluid, hatches 2 h for 37 ℃, after taking-up, wash 3 times with the phosphate buffer of 0.01M pH 7.0, room temperature is dried; The preparation of cellulose nitrate membrane closure damping fluid: 0.5%W/V PVP, 0.5%V/V Tween – 20,0.01 M pH 7.2 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions;
8) nitrocellulose filter of first step 7) being processed is pasted on the centre position of PVC base plate, then toward the end of PVC, paste successively the sample pad that PRSV gold labeling antibody pad and glass fibre element film are made, the other end pastes pastes thieving paper as the Quality Control line end;
9) after the test strips drying prepared by step 8), be the immunity colloidal gold test paper strip of PRSV, use foil sealing, 4 ℃ of preservations.
Embodiment 2
The colloid gold immune test paper bar detects the Dynamics of PRSV on cucurbita pepo
Get the Pumpkin Seedlings be incubated in the insect protected greenhouse, in wherein on 1 tender leaf, evenly sprinkling a small amount of emery, with finger, dip PRSV solution frictional inoculation on the blade face sprinkled with emery that 10 μ g/mL purify, after about 1min, with the emery on distilled water flushing inoculation leaf, postvaccinal cucurbita pepo is placed in the insect protected greenhouse and cultivates.After inoculation, on the inoculation leaf, got 0.5 g sample respectively in the 3rd, 5,7,9,11,13 and 15 days, add phosphate buffer phosphate buffer 5 mL of 0.01 M pH 7.0, fully grind, the centrifugal 1min of 5000g, supernatant are testing sample.Get respectively 200 μ L testing samples and drip on the test strips sample pad of embodiment 1 preparation, according to the colour developing situation judgement testing result of detection line on test strips and nature controlling line, adopt simultaneously indirect elisa method to detect sample after 15min, test repeats 3 times.Testing result is in Table 1, and as can be seen from the results, after Pumpkin Seedlings inoculation PRSV, the 11st talent starts reveal any symptoms, and test strips method and indirect elisa method just can detect the PRSV in blade on the 7th day after virus inoculation, than symptom time of occurrence Zao 4 days.Illustrate that the test strips of embodiment 1 preparation can be used for the early diagnosis of PRSV on cucurbita pepo, its testing result and indirect elisa method testing result are basically identical.
The detection method of reference examples ELISA is as follows:
(1) get each testing sample solution 200 μ L and add respectively in the mensuration hole of elisa plate, and respectively to add the sick plant extraction liquid for preparing by same procedure and the positive contrast in hole and the negative control of health plant extract, each processes triplicate.In 37 ℃ of water-baths, hatch 2 h or 4 ℃ of placements of spending the night.
(2) take out elisa plate, discard liquid in hole, and on thieving paper, pat dry elisa plate, every hole adds the PBST cleansing solution of 300 μ L, places 3-5 min, pats dry after washing, washs altogether 3 times.The preparation of PBST cleansing solution: 8.0g NaCl, 0.2g KH
2PO
4, 2.9g Na
2HPO
412H
2O, 0.2g KCl, 0.5 mL Tween-20, be settled to 1L with distilled water.
The PRSV polyclonal antibody that (3) will prepare, with 2000 times of PBST-PVP dilutions, adds the polyclonal antibody after 100 μ L dilute in the every hole of elisa plate, 1 h is hatched in 37 ℃ of water-baths.The preparation of PBST-PVP: add the PVP of 20 g in every liter of PBST and fully dissolve.
(4) repeating step (2).
(5) in the every hole of elisa plate, add the alkali phosphatase enzyme mark goat anti-rabbit igg of 100 μ L with 30000 times of PBST-PVP dilutions, hatch 1 h for 37 ℃.
(6) repeating step (2).
(7) in the every hole of elisa plate, add 100 μ L with the 1 p-nitrobenzophenone disodium hydrogen phosphate of mg/mL (PNPP) solution that substrate buffer solution dissolves, hatch approximately 30 min for 37 ℃.The substrate buffer solution preparation: 97 mL diethanolamine, 800 mL distilled water, be adjusted to pH9.8 with concentrated hydrochloric acid, and distilled water is settled to 1 L.
(8) take out elisa plate, on microplate reader, measure the light absorption value of each hole 405 nm.
(9) light absorption value of hole and control wells carrys out judged result per sample, and when the light absorption value of sample well was more than or equal to 2 times of negative control value, sample was judged as the positive, otherwise negative.The detectability of the method is about 0. 1 μ g/mL.
Embodiment 3
From the papaya planting site of different regions, Fujian Province, gather 50 parts, doubtful Resisting Ringspot Virus of Papaya sample, take respectively each sample 0.5 g, phosphate buffer phosphate buffer 5 mL that add 0.01 M pH 7.0, fully grind, centrifugal 1 min of 5000 g, supernatant is testing sample solution.The test strips of embodiment 1 preparation is inserted in testing sample solution, and the colour developing situation according to detection line on test strips and nature controlling line after 10 min judges testing result.Result shows in 50 duplicate samples has 46 parts to be positive, and 4 parts are negative; Adopt simultaneously indirect ELISA method respectively 50 duplicate samples to be detected, testing result has 47 parts to be positive, and 3 parts are negative.Relatively 2 kinds of detection method acquired results can be found out, the accuracy of ELISA test strip result and reliability are near the ELISA detection method.
Claims (4)
1. one kind is detected the PRSV immunity colloidal gold test paper strip, it is characterized in that the preparation method comprises the steps:
1) purification of PRSV: PRSV-Vb strain frictional inoculation is infected on host's cucurbita pepo in system, and the cucurbita pepo blade that classical symptom occurs of take is purified as the material of purification PRSV, obtains the PRSV of purification;
2) preparation of polyclonal antibody: the PRSV that the step 1) of take is purified is antigen, and Freunds adjuvant is emulsifying agent, immune new zealand white rabbit according to a conventional method, the polyclonal antibody of the anti-PRSV of preparation;
3) polyclonal antibody purifying: to step 2) prepared anti-PRSV polyclonal antibody carries out purifying;
4) preparation of colloidal gold solution: the tetra chlorauric acid solution in 100 mL 0.01% w/v prepares colloidal gold solution by the ratio of the 1% w/v sodium citrate solution reduction of 1.5 mL – 3 mL, solution stirs and boils 15 min, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions;
5) preparation of PRSV gold labeling antibody: get colloidal gold solution prepared by 100 mL step 4), use 0.1M K
2CO
3Regulate colloidal gold solution pH value to 7.2 – 8.0, then add while stirring the antibody of 1.5 mg – 2 mg step 3) purifying, stir and evenly mix, add 5% w/v bovine serum albumin to seal, addition is 1% w/v for making the bovine serum albumin final concentration; Solution after sealing in 250 *
gCentrifugal 20 min – 30 min, discard the formed precipitation of gold grain of gathering, supernatant in 14000 *
gCentrifugal 40 min – 60 min, abandoning supernatant, precipitation adds buffer suspension liquid to 100 mL and suspends; Buffer suspension liquid is: contain 1% w/vBSA and 0.02% w/v NaN
30.01 M pH 7.2-8.0 phosphate buffer, the filtrate after 0.22 μ m filtering with microporous membrane is preserved under 4 ℃; Solution after suspension repeats high speed centrifugation once, abandoning supernatant, and precipitation is suspended to 10 mL with above-mentioned same buffer suspension liquid again, and with 0.22 μ m filtering with microporous membrane, filtrate is PRSV gold labeling antibody solution, preserves under 4 ℃ of conditions;
6) preparation of PRSV gold labeling antibody pad: 4 mm that will cut * 6 mm glass fibre element films are immersed in 30 min in PRSV gold labeling antibody pad sealing damping fluid, 37 ℃ of dry 30-60 min in advance; The golden labeling antibody solution 10 – 20 μ L mixing liquids of getting after dilution are sprayed on the glass fibre element film that above-mentioned pre-service is good, and 37 ℃ of drying 30 min – 2 h, make PRSV gold labeling antibody pad; The preparation of wherein said PRSV gold labeling antibody pad sealing damping fluid: 0.2% w/v BSA, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions; The preparation of the golden labeling antibody solution after described dilution: get PRSV gold labeling antibody solution 1 mL prepared by step 5), add PRSV gold labeling antibody dilution buffer liquid 1 mL – 3 mL and mix; Described PRSV gold labeling antibody dilution buffer liquid be prepared as 1% w/v BSA, 2.5% w/v sucrose, 0.01 M pH 7.2 – 8.0 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collection filtrate is preserved under 4 ℃ of conditions;
7) making of detection line and nature controlling line: it is 1.6 – 2.0 mg/mL that the antibody after the step 3) purifying is diluted to concentration with 0.01 M pH 8.0 phosphate buffers, it is 0.5 – 0.6 mg/mL that goat anti-rabbit igg is diluted to concentration with 0.01 M pH 8.0 phosphate buffers, then the PRSV antibody after diluting and goat anti-rabbit igg are put respectively on nitrocellulose filter detection line and nature controlling line with 2.5 μ L/cm, two lines, 5 mm of being separated by, 37 ℃ of drying 30 – 60 min; Dried nitrocellulose filter is immersed in cellulose nitrate membrane closure damping fluid, hatches 1 – 2 h for 37 ℃, after taking-up, wash 3 times with the phosphate buffer of 0.01M pH 7.0, room temperature is dried; The preparation of cellulose nitrate membrane closure damping fluid: 0.5%W/V PVP, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffers, with 0.22 μ m filtering with microporous membrane, collect filtrate and preserve under 4 ℃ of conditions;
8) nitrocellulose filter of first step 7) being processed is pasted on the centre position of PVC base plate, then toward the end of PVC, paste successively the sample pad that PRSV gold labeling antibody pad and glass fibre element film are made, the other end pastes pastes thieving paper as the Quality Control line end;
9) after the test strips drying prepared by step 8), be the immunity colloidal gold test paper strip of PRSV, use foil sealing, 4 ℃ of preservations.
2. test strips as claimed in claim 1 is characterized in that the purification specific practice of described PRSV is as follows:
PRSV – Vb strain frictional inoculation is infected on host's cucurbita pepo in system, take cucurbita pepo blade that classical symptom the occurs material as purification PRSV; Get the sick leaf of 100-500 g in mortar, add liquid nitrogen to not having sick leaf, rapidly with grinding rod by sick leaf grind into powder, by powder transfer, to beaker, be the phosphate buffer A that 1:2-3 adds 4 ℃ of lower precoolings by mass volume ratio; Described phosphate buffer A concentration is 0.5M, and pH7.2 separately contains 0.1 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3%w/v Na
2SO
3, 0.5% w/v tritonX-100; Under 4 ℃, stir 1h and must be suspended damping fluid, by being suspended damping fluid: mixeding liquid volume is than for 5:3, adding CCl
4With CHCl
3The equal-volume mixed liquor, stir 30 min under 4 ℃, 3000 *
gCentrifugal 15 min, get upper solution, upper solution through 6000 *
gCentrifugal 15 min, get in supernatant C and add PEG6000 and NaCl to be respectively 6% w/v and 3% w/v to final concentration, after stirring and dissolving under 4 ℃ standing 2-10 h, 6000 *
gCentrifugal 15-30 min, go supernatant to stay precipitation, adds the phosphate buffer B of the 0.1M pH7.2 of supernatant C 1/5 volume; Described phosphate buffer B concentration is 0.1M, and pH7.2 separately contains 0.01 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3% Na
2SO
3, 0.5% tritonX-100; 4 ℃ of lower magnetic forces stir 4-8 h, 10000 *
gCentrifugal 15 min, get supernatant, in supernatant, adds PEG6000 and NaCl to be respectively 6% w/v and 3% w/v to final concentration, after stirring and dissolving under 4 ℃ standing 2-6 h, 6000 *
gCentrifugal 15 min, go supernatant to stay precipitation, adds supernatant C 1/20 V
0Phosphate buffer B, stir 1-2 h under 4 ℃, 8000 *
gCentrifugal 15 min, get supernatant, and supernatant carefully is added on the sucrose pad into 20%w/v, 110000 *
gCentrifugal 1.5-2 h, go supernatant to get precipitation, and precipitation is viral crude extract after adding the phosphate buffer of 2-4 mL0.1M pH7.2 to dissolve; In centrifuge tube, add successively each 3 mL of 30% sucrose solution that contain 0.7M, 0.5M, 0.3M and 0M cesium sulfate, standing 8-12 h in 4 ℃ of refrigerators, make the sucrose cesium sulfate solution form continuous gradient, viral crude extract be added on continuous gradient solution, immediately 4 ℃ 160000 *
gLower centrifugal 3 h, take out centrifuge tube, with electric torch, irradiates the visible glassy yellow virus of centrifuge tube band in darkroom, carefully draws the solution at viral band position, with after 1-3 mL phosphate buffer B dilution, 4 ℃ 100000 *
gLower centrifugal 1-2 h, precipitation is the PRSV of purification.
3. test strips as claimed in claim 1 is characterized in that described polyclonal antibody purifying is specially: the anti-PRSV polyclonal antibody that will prepare adopts Protein A affinity column to carry out purifying.
4. test strips as claimed in claim 1 is characterized in that usage is:
1) will treat that measuring plants gets the phosphate buffer 3-6 mL that 0.5g adds 0.01 M pH 7.0, fully grind, then centrifuging and taking supernatant, i.e. testing sample solution;
2) after the PRSV immunity colloidal gold test paper strip is recovered to room temperature, the sample pad of test strips is immersed in the testing sample solution of preparation, or in the sample pad place, drip testing sample solution prepared by about 200 μ L, react result of determination after 5 – 30 min minutes;
3) result is judged: if redness all appears in the detection line of test strips and nature controlling line place, illustrate in this sample that the content that contains PRSV and virus surpasses the detection lower limit of immunity colloidal gold test paper strip; Red stripes does not appear in the detection line place if the nature controlling line place manifests red stripes, and the content that do not contain PRSV or PRSV in this sample detection lower limit lower than immunity colloidal gold test paper strip is described; If red stripes does not all appear in detection line and nature controlling line place, illustrate that this test strips lost efficacy, measured result is unavailable.
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