CN103412121B - 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
- Publication number
- CN103412121B CN103412121B CN201310305087.9A CN201310305087A CN103412121B CN 103412121 B CN103412121 B CN 103412121B CN 201310305087 A CN201310305087 A CN 201310305087A CN 103412121 B CN103412121 B CN 103412121B
- Authority
- CN
- China
- Prior art keywords
- prsv
- solution
- phosphate buffer
- supernatant
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
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 detecting 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 causing papaya virosis.20th century forties this virus of U.S.'s reported first.China has introduced papaya in early 1950s and has carried out plant development, and nineteen fifty-nine has just found this virus on papaya, and after 1962, PRSV is widely current in China.Papaya plant is once be subject to infecting of PRSV, and mortality ratio up to 90%, can make papaya produce and be subject to destructive strike.Thousands of mu of papaya of Hainan Province's plantation in 2000 almost have no harvest because of infecting of PRSV.Due to seriously causing harm of PRSV, the multiple regional papaya of China produces the planting patterns being forced to take the spring planting autumn to cut, namely when spring by under papaya seedling kind, and time in autumn, tree is cut down, make perennial papaya people for becoming annual planting, not only give to produce and bring very large trouble, also result in 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 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 the need to special instruments and equipment, but have the following disadvantages: (1) needs to adopt a series of differential host to identify, and layman is often faced with the problem symptom occurred being difficult to accurately judgement.(2) in reality qualification, often multiple viral Combined Infection host, makes symptom produce change greatly, not easily judges; (3) identify that speed is slow, the cycle is long.After inoculation, differential host produces time needed for symptom generally more than 3 days, and what have even reaches more than 20 days; (4) qualification result is subject to the impact of season and surrounding enviroment, and the space that needs one are relatively independent, avoid the cross infection of virus; (5) some virus there is no suitable differential host, and traditional biological method cannot be adopted to identify.Though enzyme-linked immunosorbent assay and molecular biology method detect virus have the advantage that detection specificity is strong, highly sensitive, detection time is short compared with 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 through special training, these the two kinds methods detecting virus can only be completed in laboratory, be unfavorable for carrying out promoting and using in grass-roots unit; (2) compared with traditional biological method, though there is very large shortening detection time, required time at least also wants a few hours, is not still suitable for the quick detection at scene, field; (3) cost needed for detection is higher, particularly molecular biology for detection, needs to use expensive reagent.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 becomes a kind of stable colloidal state due to electrostatic interaction.Colloidal gold-labeled method is exactly the addition by regulating reductive agent, prepare the colloid gold particle of different size and color, these colloid gold particles have very strong adsorbability to albumen, can be combined to form naked eyes and can see red or pink color with immunoglobulin (Ig) non-covalent bond.The advantage of colloid gold immune test paper bar detection method is without the need to by laboratory equipment and professional and technical personnel, the water taken a morsel only is needed to drop in the sample pad in test strips, just testing result can be obtained in tens minutes even a few minutes, the feature such as have safe and simple, no radioactivity pollute, highly sensitive, result is accurate, cost is low, is 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 quick detection PRSV, solves current field field quick detection PRSV and still lacks effective means problem.Test strips prepared by the present invention have simple to operate, detect fast, result accurately and reliably, with low cost, without the need to by special instrument and equipment, be applicable to the features such as field Site Detection, can be used for the early diagnosis of field diseased plant, control for Resisting Ringspot Virus of Papaya provides effective early warning, that improves Resisting Ringspot Virus of Papaya prevents and treats efficiency, reduces the economic loss caused by it.
a kind of detection PRSV immunity colloidal gold test paper strip, is characterized in that preparation method comprises the steps:
1) purification of PRSV: by PRSV-Vb strain frictional inoculation on system infections host cucurbita pepo, to occur that the cucurbita pepo blade of classical symptom is purified for the material of purification PRSV, obtains the PRSV of purification;
2) preparation of polyclonal antibody: the PRSV purified with step 1) is for antigen, and Freund's adjuvant is emulsifying agent, according to a conventional method immunize New Zealand White Rabbit, prepares the polyclonal antibody of anti-PRSV;
3) polyclonal antibody purification: to step 2) prepared by anti-PRSV polyclonal antibody carry out purifying;
4) preparation of colloidal gold solution: prepare colloidal gold solution by the ratio of the 1% w/v sodium citrate solution reduction of 1.5 mL – 3 mL in the tetra chlorauric acid solution of 100 mL 0.01% w/v, solution stirs and boils 15 min, with 0.22 μm of filtering with microporous membrane, collect filtrate and preserve under 4 DEG C 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 the antibody of 1.5 mg – 2 mg step 3) purifying while stirring, stir and evenly mix, add 5% w/v bovine serum albumin and close, addition is 1% w/v for making bovine serum albumin final concentration; Solution after closing in 250 ×
gcentrifugal 20 min – 30 min, the precipitation that the gold grain discarding gathering is formed, supernatant in 14000 ×
gcentrifugal 40 min – 60 min, abandoning supernatant, precipitation adds buffer suspension liquid to 100 mL and suspends; Buffer suspension liquid is: containing 1% w/vBSA and 0.02% w/v NaN
30.01 M pH 7.2-8.0 phosphate buffer, the filtrate after 0.22 μm of filtering with microporous membrane is preserved at 4 DEG C; 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 μm of filtering with microporous membrane, filtrate is PRSV gold labeling antibody solution, preserves under 4 DEG C of conditions;
6) preparation of PRSV gold labeling antibody pad: the 4 mm × 6 mm glass fibre element films cut are immersed in 30 min, 37 DEG C of dry 30-60 min in PRSV gold labeling antibody pad Block buffer in advance; Getting the golden labeling antibody solution 10 – 20 μ L mixing liquid after dilution is sprayed on the good glass fibre element film of above-mentioned pre-service, and 37 DEG C of drying 30 min – 2 h, make PRSV gold labeling antibody pad; The preparation of wherein said PRSV gold labeling antibody pad Block buffer: 0.2% w/v BSA, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collects filtrate and preserves under 4 DEG C 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), adds PRSV gold labeling antibody dilution buffer 1 mL – 3 mL mixing; Described PRSV gold labeling antibody dilution buffer be prepared as 1% w/v BSA, 2.5% w/v sucrose, 0.01 M pH 7.2 – 8.0 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collection filtrate is preserved under 4 DEG C of conditions;
7) making of detection line and nature controlling line: it is 1.6 – 2.0 mg/mL that the antibody after step 3) purifying is diluted to concentration with 0.01 M pH 8.0 phosphate buffer, 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 buffer, then the PRSV antibody after dilution and goat anti-rabbit igg are put on nitrocellulose filter detection line and nature controlling line respectively with 2.5 μ L/cm, two lines are separated by 5 mm, 37 DEG C of drying 30 – 60 min; Be immersed in by dried nitrocellulose filter in nitrocellulose filter Block buffer, hatch 1 – 2 h for 37 DEG C, after taking-up, wash 3 times with the phosphate buffer of 0.01M pH 7.0, room temperature is dried; The preparation of nitrocellulose filter Block buffer: 0.5%W/V PVP, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collect filtrate and preserve under 4 DEG C of conditions;
8) first the nitrocellulose filter of step 7) process is pasted on the centre position of PVC base plate, then paste the sample pad that PRSV gold labeling antibody pad and glass fibre element film are made successively toward one end of PVC, the other end pastes pastes thieving paper as Quality Control line end;
9) immunity colloidal gold test paper strip of PRSV is after test strips drying step 8) prepared, by foil sealing, 4 DEG C of preservations.
The purification specific practice of described PRSV is as follows:
By PRSV – Vb strain frictional inoculation on system infections host cucurbita pepo, there is the material of cucurbita pepo blade for purification PRSV of classical symptom; The sick leaf getting 100-500 g, in mortar, adds liquid nitrogen to not having sick leaf, rapidly with grinding rod by sick leaf grind into powder, by powder transfer in beaker, be the phosphate buffer A that 1:2-3 adds precooling at 4 DEG C by mass volume ratio; Described phosphate buffer A concentration is 0.5M, pH7.2, another containing 0.1 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3%w/v Na
2sO
3, 0.5% w/v tritonX-100; At 4 DEG C, stir 1h must be suspended damping fluid, by being suspended damping fluid: mixeding liquid volume adds CCl than for 5:3
4with CHCl
3equal-volume mixed liquor, stir 30 min at 4 DEG C, 3000 ×
gcentrifugal 15 min, get upper solution, upper solution through 6000 ×
gcentrifugal 15 min, get in supernatant C and add PEG6000 and NaCl and be respectively 6% w/v and 3% w/v to final concentration, leave standstill 2-10 h after stirring and dissolving at 4 DEG C, 6000 ×
gcentrifugal 15-30 min, goes 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, pH7.2, another containing 0.01 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3% Na
2sO
3, 0.5% tritonX-100; 4 DEG C of lower magnetic forces stir 4-8 h, 10000 ×
gcentrifugal 15 min, get supernatant, add PEG6000 and NaCl and be respectively 6% w/v and 3% w/v to final concentration in supernatant, leave standstill 2-6 h after stirring and dissolving at 4 DEG C, 6000 ×
gcentrifugal 15 min, go supernatant to stay precipitation, add supernatant C 1/20 V
0phosphate buffer B, stir 1-2 h at 4 DEG C, 8000 ×
gcentrifugal 15 min, get supernatant, and supernatant is carefully added on the sucrose cushions into 20%w/v, 110000 ×
gcentrifugal 1.5-2 h, goes supernatant to get precipitation, and precipitation is viral crude extract after adding the phosphate buffer dissolving of 2-4 mL0.1M pH7.2; Each 3 mL of 30% sucrose solution containing 0.7M, 0.5M, 0.3M and 0M cesium sulfate are added successively in centrifuge tube, 8-12 h is left standstill in 4 DEG C of refrigerators, make sucrose cesium sulfate solution formed continuous gradient, viral crude extract is added on continuous gradient solution, immediately 4 DEG C 160000 ×
glower centrifugal 3 h, take out centrifuge tube, in darkroom, irradiate the visible glassy yellow virus band of centrifuge tube with electric torch, the careful solution drawing virus band position, after diluting with 1-3 mL phosphate buffer B, 4 DEG C 100000 ×
glower centrifugal 1-2 h, precipitation is the PRSV of purification.
Described polyclonal antibody purification is specially: adopt Protein A affinity column to carry out purifying the anti-PRSV polyclonal antibody of preparation.Thermo company can be selected
protein A Columns, and carry out purifying by its instructions purification step;
Described test strips, is characterized in that usage is:
1) the phosphate buffer 3-6 mL that measuring plants is got 0.5g and added 0.01 M pH 7.0 will be treated, fully grind, then centrifuging and taking supernatant, i.e. testing sample solution;
2) after PRSV immunity colloidal gold test paper strip being recovered room temperature, the sample pad of test strips is immersed in the testing sample solution of preparation, or drip the testing sample solution prepared of about 200 μ L in sample pad place, react 5 – 30 result of determination after min minute;
Result judges: if the detection line of test strips and nature controlling line place all occur redness, then the Monitoring lower-cut exceeding immunity colloidal gold test paper strip in this sample containing PRSV and the content of virus is described; If nature controlling line place manifests red stripes and red stripes does not appear in detection line place, the Monitoring lower-cut of content lower than immunity colloidal gold test paper strip not containing PRSV or PRSV in this sample is described; If detection line and nature controlling line place all do not occur red stripes, then illustrate that this test strips lost efficacy, measured result is unavailable.
notable feature of the present invention is: the test strips prepared by technology of the present invention can carry out quick diagnosis to the PRSV in sample in 5-20 min, this test strips low production cost, easy and simple to handle, detection speed is fast, result accurately and reliably, can field Site Detection, special instrument and equipment, operating personnel are not needed or not through special training, be easy to promote the use of, early diagnosis can be carried out to field diseased plant, reduce field poison source quantity, reduce because PRSV is caused harm the economic loss caused.
Accompanying drawing explanation
Fig. 1 is schematic diagram of the present invention
1: sample pad 2:PRSV gold labeling antibody pad 3: the nitrocellulose filter 4:PRSV polyclonal antibody point made of glass fibre element film is on the detection line of nitrocellulose filter 5: goat anti-rabbit igg point 6:PVC base plate 7 on nitrocellulose filter nature controlling line: thieving paper
Fig. 2 testing result schematic diagram
Be followed successively by from left to right: testing result is positive, and detection line (T) and nature controlling line (C) two lines all develop the color; Testing result is negative, and nature controlling line (C) line develops the color, 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
More being convenient to make content of the present invention understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention being not limited only to this.
Embodiment 1
A kind of detection PRSV immunity colloidal gold test paper strip, is characterized in that preparation method comprises the steps:
1) by PRSV – Vb strain frictional inoculation on system infections host cucurbita pepo, there is the material of cucurbita pepo blade for purification PRSV of classical symptom; The sick leaf getting 200 g, in mortar, adds liquid nitrogen to not having sick leaf, rapidly with grinding rod by sick leaf grind into powder, by powder transfer in beaker, add the phosphate buffer A of precooling at 500 mL 4 DEG C; Described phosphate buffer A concentration is 0.5M, pH7.2, another containing 0.1 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3%w/v Na
2sO
3, 0.5% w/v tritonX-100; At 4 DEG C, stir 1h must be suspended damping fluid, add 300 mL CCl
4with CHCl
3equal-volume mixed liquor, stir 30 min at 4 DEG C, 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, leave standstill 8 h after stirring and dissolving at 4 DEG C, 6000 ×
gcentrifugal 15 min, go supernatant to stay precipitation, add the phosphate buffer B of the 0.1M pH7.2 of 80 mL in supernatant; Described phosphate buffer B concentration is 0.1M, pH7.2, another containing 0.01 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3% Na
2sO
3, 0.5% tritonX-100; 4 DEG C of lower magnetic forces stir 4 h, 10000 ×
gcentrifugal 15 min, get supernatant, add the PEG6000 of 24 g and the NaCl of 12 g in supernatant, leave standstill 2 h after stirring and dissolving at 4 DEG C, 6000 ×
gcentrifugal 15 min, go supernatant to stay precipitation, add the phosphate buffer B of supernatant 20 mL, stir 1 h at 4 DEG C, 8000 ×
gcentrifugal 15 min, get supernatant, and supernatant is carefully added on the sucrose cushions 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 dissolving of 3 mL0.1M pH7.2; Each 3 mL of 30% sucrose solution containing 0.7M, 0.5M, 0.3M and 0M cesium sulfate are added successively in centrifuge tube, 12 h are left standstill in 4 DEG C of refrigerators, make sucrose cesium sulfate solution formed continuous gradient, viral crude extract is added on continuous gradient solution, immediately 4 DEG C 160000 ×
glower centrifugal 3 h, take out centrifuge tube, in darkroom, irradiate the visible glassy yellow virus band of centrifuge tube with electric torch, the careful solution drawing virus band position, after diluting with 2 mL phosphate buffer B, 4 DEG C 100000 ×
glower centrifugal 1.5 h, precipitation is the PRSV of purification.
2) preparation of polyclonal antibody: it is 2 mg/ mL that the phosphate buffer of PRSV 0.1M pH7.2 step 1) purified is diluted to concentration, get the PRSV after 1 mL dilution, add 1 mL complete Freund's adjuvant, fully emulsified rear intramuscular injection new zealand white rabbit, 10 d after first time injection, 17 d, 24 d and 31 d inject the emulsion of the PRSV after 1 mL dilution and 1 mL incomplete Freund's adjuvant respectively, after last injection, 10 d get rabbit blood, blood is placed in 4 DEG C of refrigerators, after serum is separated out, suck the polyclonal antibody that serum is preparation,
3) polyclonal antibody purification: by step 2) the anti-PRSV polyclonal antibody prepared is by Thermo company
protein A Columns instructions purification step carries out purifying;
4) preparation of colloidal gold solution: prepare colloidal gold solution by the ratio of the 1% w/v sodium citrate solution reduction of 2 mL in the tetra chlorauric acid solution of 100 mL 0.01% w/v, solution stirs and boils 15 min, with 0.22 μm of filtering with microporous membrane, collect filtrate and preserve under 4 DEG C 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 the antibody of 1.8 mg step 3) purifying while stirring, stir and evenly mix, add 5% w/v bovine serum albumin and close, addition is 1% w/v for making bovine serum albumin final concentration; Solution after closing in 250 ×
gcentrifugal 20 min, the precipitation that the gold grain discarding gathering is formed, supernatant in 14000 ×
gcentrifugal 50 min, abandoning supernatant, precipitation adds buffer suspension liquid to 100 mL and suspends; Buffer suspension liquid is: containing 1% w/vBSA and 0.02% w/v NaN
30.01 M pH 7.2 phosphate buffer, the filtrate after 0.22 μm of filtering with microporous membrane is preserved at 4 DEG C; 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 μm of filtering with microporous membrane, filtrate is PRSV gold labeling antibody solution, preserves under 4 DEG C of conditions;
6) preparation of PRSV gold labeling antibody pad: the 4 mm × 6 mm glass fibre element films cut are immersed in 30 min, 37 DEG C of drying 30 min in PRSV gold labeling antibody pad Block buffer in advance; Getting the golden labeling antibody solution 15 μ L mixing liquid after dilution is sprayed on the good glass fibre element film of above-mentioned pre-service, and 37 DEG C of dry 1h, make PRSV gold labeling antibody pad; The preparation of wherein said PRSV gold labeling antibody pad Block buffer: 0.2% w/v BSA, 0.5%V/V Tween – 20,0.01 M pH 7.6 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collects filtrate and preserves under 4 DEG C 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), adds PRSV gold labeling antibody dilution buffer 3 mL mixing; Described PRSV gold labeling antibody dilution buffer be prepared as 1% w/v BSA, 2.5% w/v sucrose, 0.01 M pH 7.6 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collection filtrate is preserved under 4 DEG C of conditions;
7) making of detection line and nature controlling line: it is 1.6 mg/mL that the antibody after step 3) purifying is diluted to concentration with 0.01 M pH 8.0 phosphate buffer, it is 0.5 mg/mL that goat anti-rabbit igg is diluted to concentration with 0.01 M pH 8.0 phosphate buffer, then the PRSV antibody after dilution and goat anti-rabbit igg are put on nitrocellulose filter detection line and nature controlling line respectively with 2.5 μ L/cm, two lines are separated by 5 mm, 37 DEG C of drying 60 min; Be immersed in by dried nitrocellulose filter in nitrocellulose filter Block buffer, hatch 2 h for 37 DEG C, after taking-up, wash 3 times with the phosphate buffer of 0.01M pH 7.0, room temperature is dried; The preparation of nitrocellulose filter Block buffer: 0.5%W/V PVP, 0.5%V/V Tween – 20,0.01 M pH 7.2 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collect filtrate and preserve under 4 DEG C of conditions;
8) first the nitrocellulose filter of step 7) process is pasted on the centre position of PVC base plate, then paste the sample pad that PRSV gold labeling antibody pad and glass fibre element film are made successively toward one end of PVC, the other end pastes pastes thieving paper as Quality Control line end;
9) immunity colloidal gold test paper strip of PRSV is after test strips drying step 8) prepared, by foil sealing, 4 DEG C of preservations.
Embodiment 2
Colloid gold immune test paper bar detects the Dynamics of PRSV on cucurbita pepo
Get the Pumpkin Seedlings be incubated in insect protected greenhouse, a small amount of emery is evenly sprinkled on wherein 1 tender leaf, PRSV solution that 10 μ g/mL purify is dipped sprinkled with frictional inoculation on the blade face of emery with finger, with the emery on distilled water flushing inoculation leaf after about 1min, postvaccinal cucurbita pepo is placed in insect protected temperature indoor culture.Within after inoculation the 3rd, 5,7,9,11,13 and 15 day, get 0.5 g sample respectively on inoculation leaf, add phosphate buffer phosphate buffer 5 mL of 0.01 M pH 7.0, fully grind, the centrifugal 1min of 5000g, supernatant is testing sample.Getting 200 μ L testing samples respectively drips in the test strips sample pad of embodiment 1 preparation, judges testing result after 15min according to the colour developing situation of detection line in test strips and nature controlling line, adopts indirect elisa method to detect sample simultaneously, test repetition 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 for the 7th day after virus inoculation, more Zao than symptom time of occurrence 4 days.Illustrate that test strips prepared by embodiment 1 can be used for the early diagnosis of PRSV on cucurbita pepo, its testing result and indirect elisa method testing result basically identical.
The detection method of reference examples ELISA is as follows:
(1) get each testing sample solution 200 μ L to add respectively in the mensuration hole of elisa plate, and respectively to add the hole of sick plant extraction liquid and the health plant extract prepared by same procedure for positive control and negative control, each process in triplicate.In 37 DEG C of water-baths, hatch 2 h or 4 DEG C to spend the night placement.
(2) take out elisa plate, discard liquid in hole, and pat dry elisa plate on thieving paper, every hole adds the PBST cleansing solution of 300 μ L, places 3-5 min, pats dry after washing, washs 3 times altogether.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, is settled to 1L with distilled water.
(3) the PRSV polyclonal antibody PBST-PVP of preparation is diluted 2000 times, add the polyclonal antibody after 100 μ L dilutions in the every hole of elisa plate, 1 h is hatched in 37 DEG C of water-baths.The preparation of PBST-PVP: add the PVP of 20 g in often liter of PBST and fully dissolve.
(4) step (2) is repeated.
(5) add the alkali phosphatase enzyme mark goat anti-rabbit igg that 100 μ L PBST-PVP dilute 30000 times in the every hole of elisa plate, hatch 1 h for 37 DEG C.
(6) step (2) is repeated.
(7) add 1 mg/mL p-nitrophenylphosphate disodium (PNPP) solution that 100 μ L substrate buffer solutions dissolve in the every hole of elisa plate, hatch about 30 min for 37 DEG C.Substrate buffer solution is prepared: 97 mL diethanolamine, 800 mL distilled water, are adjusted to pH9.8 with concentrated hydrochloric acid, and distilled water is settled to 1 L.
(8) take out elisa plate, microplate reader measures 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 is more than or equal to 2 times of negative control value, sample is judged as the positive, otherwise is negative.The detectability of the method is about 0. 1 μ g/mL.
Embodiment 3
50 parts, doubtful Resisting Ringspot Virus of Papaya sample is gathered from the papaya planting site of different regions, Fujian Province, take each sample 0.5 g respectively, add phosphate buffer phosphate buffer 5 mL of 0.01 M pH 7.0, abundant grinding, centrifugal 1 min of 5000 g, supernatant is testing sample solution.Test strips embodiment 1 prepared is inserted in testing sample solution, judges testing result after 10 min according to the colour developing situation of detection line in test strips and nature controlling line.Result shows have 46 parts to be positive in 50 increment product, and 4 parts are negative; Adopt indirect ELISA method to detect 50 increment product respectively, testing result has 47 parts to be positive, and 3 parts are negative simultaneously.Relatively 2 kinds of detection method acquired results can be found out, the accuracy of ELISA test strip result and reliability are close to ELISA detection method.
Claims (3)
1. detect a PRSV immunity colloidal gold test paper strip, it is characterized in that preparation method comprises the steps:
1) purification of PRSV: by PRSV-Vb strain frictional inoculation on system infections host cucurbita pepo, to occur that the cucurbita pepo blade of classical symptom is purified for the material of purification PRSV, obtains the PRSV of purification;
2) preparation of polyclonal antibody: the PRSV purified with step 1) is for antigen, and Freund's adjuvant is emulsifying agent, according to a conventional method immunize New Zealand White Rabbit, prepares the polyclonal antibody of anti-PRSV;
3) polyclonal antibody purification: to step 2) prepared by anti-PRSV polyclonal antibody carry out purifying;
4) preparation of colloidal gold solution: prepare colloidal gold solution by the ratio of the 1% w/v sodium citrate solution reduction of 1.5 mL – 3 mL in the tetra chlorauric acid solution of 100 mL 0.01% w/v, solution stirs and boils 15 min, with 0.22 μm of filtering with microporous membrane, collect filtrate and preserve under 4 DEG C 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 the antibody of 1.5 mg – 2 mg step 3) purifying while stirring, stir and evenly mix, add 5% w/v bovine serum albumin and close, addition is 1% w/v for making bovine serum albumin final concentration; Solution after closing in 250 ×
gcentrifugal 20 min – 30 min, the precipitation that the gold grain discarding gathering is formed, supernatant in 14000 ×
gcentrifugal 40 min – 60 min, abandoning supernatant, precipitation adds buffer suspension liquid to 100 mL and suspends; Buffer suspension liquid is: containing 1% w/vBSA and 0.02% w/v NaN
30.01 M pH 7.2-8.0 phosphate buffer, the filtrate after 0.22 μm of filtering with microporous membrane is preserved at 4 DEG C; 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 μm of filtering with microporous membrane, filtrate is PRSV gold labeling antibody solution, preserves under 4 DEG C of conditions;
6) preparation of PRSV gold labeling antibody pad: the 4 mm × 6 mm glass fibre element films cut are immersed in 30 min, 37 DEG C of dry 30-60 min in PRSV gold labeling antibody pad Block buffer in advance; Getting the golden labeling antibody solution 10 – 20 μ L mixing liquid after dilution is sprayed on the good glass fibre element film of above-mentioned pre-service, and 37 DEG C of drying 30 min – 2 h, make PRSV gold labeling antibody pad; The preparation of wherein said PRSV gold labeling antibody pad Block buffer: 0.2% w/v BSA, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collects filtrate and preserves under 4 DEG C 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), adds PRSV gold labeling antibody dilution buffer 1 mL – 3 mL mixing; Described PRSV gold labeling antibody dilution buffer be prepared as 1% w/v BSA, 2.5% w/v sucrose, 0.01 M pH 7.2 – 8.0 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collection filtrate is preserved under 4 DEG C of conditions;
7) making of detection line and nature controlling line: it is 1.6 – 2.0 mg/mL that the antibody after step 3) purifying is diluted to concentration with 0.01 M pH 8.0 phosphate buffer, 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 buffer, then the PRSV antibody after dilution and goat anti-rabbit igg are put on nitrocellulose filter detection line and nature controlling line respectively with 2.5 μ L/cm, two lines are separated by 5 mm, 37 DEG C of drying 30 – 60 min; Be immersed in by dried nitrocellulose filter in nitrocellulose filter Block buffer, hatch 1 – 2 h for 37 DEG C, after taking-up, wash 3 times with the phosphate buffer of 0.01M pH 7.0, room temperature is dried; The preparation of nitrocellulose filter Block buffer: 0.5%W/V PVP, 0.5%V/V Tween – 20,0.01 M pH 7.2 – 8.0 phosphate buffer, with 0.22 μm of filtering with microporous membrane, collect filtrate and preserve under 4 DEG C of conditions;
8) first the nitrocellulose filter of step 7) process is pasted on the centre position of PVC base plate, then paste the sample pad that PRSV gold labeling antibody pad and glass fibre element film are made successively toward one end of PVC, the other end pastes pastes thieving paper as Quality Control line end;
9) immunity colloidal gold test paper strip of PRSV is after test strips drying step 8) prepared, by foil sealing, 4 DEG C of preservations;
The purification specific practice of described PRSV is as follows:
By PRSV – Vb strain frictional inoculation on system infections host cucurbita pepo, there is the material of cucurbita pepo blade for purification PRSV of classical symptom; The sick leaf getting 100-500 g, in mortar, adds liquid nitrogen to not having sick leaf, rapidly with grinding rod by sick leaf grind into powder, by powder transfer in beaker, be the phosphate buffer A that 1:2-3 adds precooling at 4 DEG C by mass volume ratio; Described phosphate buffer A concentration is 0.5M, pH7.2, another containing 0.1 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3%w/v Na
2sO
3, 0.5% w/v tritonX-100; At 4 DEG C, stir 1h must be suspended damping fluid, by being suspended damping fluid: mixeding liquid volume adds CCl than for 5:3
4with CHCl
3equal-volume mixed liquor, stir 30 min at 4 DEG C, 3000 ×
gcentrifugal 15 min, get upper solution, upper solution through 6000 ×
gcentrifugal 15 min, get in supernatant C and add PEG6000 and NaCl and be respectively 6% w/v and 3% w/v to final concentration, leave standstill 2-10 h after stirring and dissolving at 4 DEG C, 6000 ×
gcentrifugal 15-30 min, goes 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, pH7.2, another containing 0.01 M disodium ethylene diamine tetraacetate, 1 M urea, 0.3% Na
2sO
3, 0.5% tritonX-100; 4 DEG C of lower magnetic forces stir 4-8 h, 10000 ×
gcentrifugal 15 min, get supernatant, add PEG6000 and NaCl and be respectively 6% w/v and 3% w/v to final concentration in supernatant, leave standstill 2-6 h after stirring and dissolving at 4 DEG C, 6000 ×
gcentrifugal 15 min, go supernatant to stay precipitation, add the phosphate buffer B of supernatant C 1/20 volume, stir 1-2 h at 4 DEG C, 8000 ×
gcentrifugal 15 min, get supernatant, and supernatant is carefully added on the sucrose cushions into 20%w/v, 110000 ×
gcentrifugal 1.5-2 h, goes supernatant to get precipitation, and precipitation is viral crude extract after adding the phosphate buffer dissolving of 2-4 mL0.1M pH7.2; Each 3 mL of 30% sucrose solution containing 0.7M, 0.5M, 0.3M and 0M cesium sulfate are added successively in centrifuge tube, 8-12 h is left standstill in 4 DEG C of refrigerators, make sucrose cesium sulfate solution formed continuous gradient, viral crude extract is added on continuous gradient solution, immediately 4 DEG C 160000 ×
glower centrifugal 3 h, take out centrifuge tube, in darkroom, irradiate the visible glassy yellow virus band of centrifuge tube with electric torch, the careful solution drawing virus band position, after diluting with 1-3 mL phosphate buffer B, 4 DEG C 100000 ×
glower centrifugal 1-2 h, precipitation is the PRSV of purification.
2. test strips as claimed in claim 1, is characterized in that described polyclonal antibody purification is specially: adopt Protein A affinity column to carry out purifying the anti-PRSV polyclonal antibody of preparation.
3. test strips as claimed in claim 1, is characterized in that usage is:
1) the phosphate buffer 3-6 mL that measuring plants is got 0.5g and added 0.01 M pH 7.0 will be treated, fully grind, then centrifuging and taking supernatant, i.e. testing sample solution;
2) PRSV immunity colloidal gold test paper strip is recovered after room temperature, the sample pad of test strips is immersed in the testing sample solution of preparation, or drip testing sample solution prepared by 200 μ L in sample pad place, react 5 – 30 result of determination after min minute;
3) result judges: if the detection line of test strips and nature controlling line place all occur redness, then the Monitoring lower-cut exceeding immunity colloidal gold test paper strip in this sample containing PRSV and the content of virus is described; If nature controlling line place manifests red stripes and red stripes does not appear in detection line place, the Monitoring lower-cut of content lower than immunity colloidal gold test paper strip not containing PRSV or PRSV in this sample is described; If detection line and nature controlling line place all do not occur red stripes, then illustrate that this test strips lost efficacy, measured result is unavailable.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310305087.9A CN103412121B (en) | 2013-07-20 | 2013-07-20 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310305087.9A CN103412121B (en) | 2013-07-20 | 2013-07-20 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103412121A CN103412121A (en) | 2013-11-27 |
| CN103412121B true CN103412121B (en) | 2015-02-04 |
Family
ID=49605147
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310305087.9A Expired - Fee Related CN103412121B (en) | 2013-07-20 | 2013-07-20 | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103412121B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107907680B (en) * | 2017-09-30 | 2020-02-18 | 伊犁出入境检验检疫局综合技术服务中心 | Colloidal gold immune test paper for detecting bacillus larvae of bees and preparation and application thereof |
| CN109870575A (en) * | 2017-12-04 | 2019-06-11 | 厦门华侨亚热带植物引种园 | A method of improving papaya ringspot virus antibody specificity |
| CN113668069B (en) * | 2020-05-13 | 2023-12-08 | 洛阳中科生物芯片技术有限公司 | Preparation method of protein chip board |
| CN116773802A (en) * | 2023-08-18 | 2023-09-19 | 南京市产品质量监督检验院(南京市质量发展与先进技术应用研究院) | Test strip for detecting wheat stinking smut by using colloidal gold immunochromatography |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101246172A (en) * | 2007-02-14 | 2008-08-20 | 北京华安佛医药研究中心有限公司 | Folic acid immunity colloidal gold detecting test paper and method for producing the same |
| CN101294962A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
| CN101294963A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
| CN101881770A (en) * | 2009-05-08 | 2010-11-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN101968488A (en) * | 2010-10-26 | 2011-02-09 | 吉林大学 | Colloidal gold fast diagnostic test strip of goose paramyxovirus disease |
| CN102020714A (en) * | 2010-09-26 | 2011-04-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody |
| KR20130054482A (en) * | 2011-11-11 | 2013-05-27 | 한국생명공학연구원 | Probe set for diagnosing or detecting virus of genus potyvirus and uses thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040241776A1 (en) * | 2003-05-22 | 2004-12-02 | Agdia, Inc. | Multiplex enzyme-linked immunosorbent assay for detecting multiple analytes |
| CN102277376B (en) * | 2010-06-10 | 2013-09-25 | 叶锡东 | Gene transfer vector providing crop broad-spectrum virus resistance and containing pawpaw ringspot virus helper component protease gene and application thereof |
| CN102899335A (en) * | 2012-09-14 | 2013-01-30 | 中国热带农业科学院热带生物技术研究所 | Method for obtaining genome sequence of papaya ringspot virus through high-throughput small RNA sequencing |
-
2013
- 2013-07-20 CN CN201310305087.9A patent/CN103412121B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101246172A (en) * | 2007-02-14 | 2008-08-20 | 北京华安佛医药研究中心有限公司 | Folic acid immunity colloidal gold detecting test paper and method for producing the same |
| CN101294962A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
| CN101294963A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
| CN101881770A (en) * | 2009-05-08 | 2010-11-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN102020714A (en) * | 2010-09-26 | 2011-04-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Colloidal gold test paper for jointly detecting abrin and ricin and special monoclonal antibody |
| CN101968488A (en) * | 2010-10-26 | 2011-02-09 | 吉林大学 | Colloidal gold fast diagnostic test strip of goose paramyxovirus disease |
| KR20130054482A (en) * | 2011-11-11 | 2013-05-27 | 한국생명공학연구원 | Probe set for diagnosing or detecting virus of genus potyvirus and uses thereof |
Non-Patent Citations (5)
| Title |
|---|
| Purification and Serology of Papaya Ringspot Virus;D.Gonsalves,et al.;《Phytopathology》;19801231;第70卷(第11期);第1028-1032页 * |
| 植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术;肖火根 等;《中国病毒学》;19940930;第9卷(第3期);摘要，第249-250页"材料与方法"部分 * |
| 番木瓜环斑病毒外壳蛋白基因原核表达蛋白的抗血清制备及其检测应用;赵芹 等;《园艺学报》;20121231;第39卷(第8期);第1457-1464页 * |
| 番木瓜环斑病毒外壳蛋白基因的cDNA克隆及全序列测定比较;龚洁敏 等;《植物学报》;19931231;第35卷(第6期);第416-421页 * |
| 番木瓜环斑病毒检测技术的研究;陈枝楠 等;《中国病毒学》;19930630;第8卷(第2期);第165-166页"二、方法"部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103412121A (en) | 2013-11-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110007096A (en) | A kind of dengue virus IgG/IgM antibody test strip, kit and preparation method thereof | |
| CN103412121B (en) | Colloidal gold immune test strip for rapid detection of Papaya ringspot virus | |
| CN107942061A (en) | A kind of test card, preparation and its detection method for detecting transmissible gastro-enteritis virus antibody | |
| CN104181298B (en) | A kind of human parainfluenza virus's IgM antibody test strip and preparation method thereof | |
| CN110007095A (en) | A kind of dengue virus NS 1 antigen test strip, kit and preparation method thereof | |
| CN104122389A (en) | Reagent card for colloidal gold immunochromatograohic assay of lily mottle virus and preparation method of reagent card for colloidal gold immunochromatograohic assay of lily mottle virus | |
| CN102879561B (en) | Screening test paper strip for human lactoferrin in milk and preparation method | |
| CN103954772B (en) | Three gold mark detection test paper bars of a kind of CSFV, pig breeding and respiratory disorder syndrome virus, Pseudorabies virus antibody | |
| CN102109526B (en) | Test paper for quickly detecting grass carp reovirus (GCRV) and preparation method and using method thereof | |
| CN102809653B (en) | Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen | |
| CN104730248A (en) | Rapid diagnosis and detection test paper card for detecting swine fever as well as preparation and application method of rapid diagnosis and detection test paper card | |
| CN202599960U (en) | Colloidal gold test strip for rapid detection of O-type foot-and-mouth disease virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus | |
| CN205246668U (en) | Quick detect reagent strip of vibrio alginolyticus | |
| CN104122391B (en) | A kind of hidden syndrome virus of lily and lily mottle virus composite colloid gold immunochromatographiassay assay reagent card and preparation method | |
| CN105585633B (en) | The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application antibody | |
| CN104122390B (en) | Reagent card for detecting lily symptomless virus by adopting colloidal gold immunochromatographic assay and preparation method | |
| CN101000345B (en) | Immunological chromatographic test paper for testing and its preparation method | |
| CN105585635B (en) | The immune chromatography reagent kit of anti-human mycoplasma pneumoniae p1 protein antibody and the application antibody | |
| CN1632585A (en) | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof | |
| CN102305861B (en) | Reagent strip for carrying out joint detection on Toxoplasma gondii IgM (immunoglobulin M) and IgG (immunoglobulin G) antibodies and preparation method thereof | |
| CN102360012A (en) | Immunochromatography detection reagent strip for combined detection of toxoplasmagondii IgG antibodies and total antibodies, and preparation method thereof | |
| CN202256347U (en) | Reagent strips for joint detection of toxoplasma immunoglobulin M (IgM) antibody and total antibody | |
| CN102621319B (en) | Colloidal gold quick diagnosis test paper for distinguishing classic porcine reproductive and respiratory syndrome (PRRS) from highly pathogenic PRRS (HPRRS) | |
| CN102393454A (en) | Colloidal gold test strip for detecting dengue virus antibody and preparation method and application thereof | |
| CN101852802A (en) | Test paper for detecting newcastle disease and infectious bronchitis viruses in one step |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150204 Termination date: 20180720 |