CN1869698A - Preparation technical method of potato PVX, PVY< PLRV, PVS virus diagnostic reagent - Google Patents
Preparation technical method of potato PVX, PVY< PLRV, PVS virus diagnostic reagent Download PDFInfo
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- CN1869698A CN1869698A CN 200410044129 CN200410044129A CN1869698A CN 1869698 A CN1869698 A CN 1869698A CN 200410044129 CN200410044129 CN 200410044129 CN 200410044129 A CN200410044129 A CN 200410044129A CN 1869698 A CN1869698 A CN 1869698A
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Abstract
The invention relates to a reagent, belonging to a process for making a reagent for diagnosing potato PVX, PVY, PLRV, and PVS viruses, characterized in that it collects materials with typical potato virus symptoms from potato production filed, screening the needed virus source materials by ELISA identification, selecting the materials with positive identification results and higher light absorption values, then using transmitting electronic microscope (TEM) to make auxiliary check and determining the virus source materials; inoculating a series of healthy indication plants planted in greenhouse with the virus source materials and making virus separation research, and investigating the inoculated plants once every three days and according to the disease conditions of each group of indication plants, screening and separating out the needed viruses, then making multiple inoculation to filter out the other viruses, making virus purification, then inoculating virus breeding material with the purified virus for purification.
Description
Technical field: the present invention relates to reagent, belong to potato PVX, PVY, PLRV, PVS virus diagnose reagent process for making.
Background technology: potato is the main grain of China, dish crop and raw materials of food processing, and 4,600,000 hectares of national cultivated areas occupy first place in the world.But yield level lower (11 tons/hectare) not only is lower than world's per unit area yield (15.5 tons/hectare), more too late world developed country (30-50 ton/hectare).Influence the principal element of China's potato yield, it is virus disease, plant virus is an obligate parasite, can only in its host's living cells, duplicate, potato is vegetative reproduction, accumulation year by year causes the potato seed sexual involution to virus with the potato breeding, studies have shown that Potyvirus causes that the potato underproduction is at 30-35%.At present, have more than 25 kinds in virus that has been found that on the potato crop and report and virosis, fortunately have only minority virus heavier to potato harm, compound infecting than a certain virus infected separately seriously usually, and it is compound infecting that potato produces Tanaka most.The present invention is directed to potato produce in heavier viral PVX, PVY, PVS and the PLRV of harm study.
Be to utilize the immune serum technology to eliminate to Potyvirus control optimum method in the world at present,, produce the toxicity-removing white potato potato seed in conjunction with stem apex detoxify, tissue culture except that diseased plant.Enzyme connection (DAS-ELISA) method detects Potyvirus, because of its have fast, accurately, sensitive characteristics is international, domestic generally employing.
Along with China joined WTO, homemade toxicity-removing white potato production is on the hazard, plant quarantine and virus detect must be in line with international standards, 4,600,000 hectares of whole nation potato culture areas, 30.7 ten thousand hectares in potato seed field, detect 200 samples by 1 hectare of standardized production requirement and calculate, the whole nation is total to 6,000 ten thousand of samples, and year needs 600,000 of kits.After entering WTO, therefore the enforcement of China's strategy to develop western regions in addition be badly in need of large batch of high-quality, the homemade Potyvirus antiserum of low price.Serum virus is the key content during the enzyme joint inspection is surveyed, utilize immunological technique to prepare the Potyvirus height existing several years history of serum detection kit of tiring in the world, but can produce a few countries of having only of high-quality Potyvirus serum in batches, so price is higher, for example global maximum plant disease diagnostic reagent manufacturer---4200 yuan of every virus 5 00 reacting holes of U.S. agdia company; 4000 yuan of every virus 5 00 reacting holes of Germany Loewe Biochem Technology, INC. ... China has just begun The Research of Relevant Technology as far back as the eighties in 20th century, but there is the reagent poor specificity, the not congruent problems of testing process complexity, kind, the exploitation of always failing to produce in batches.
Summary of the invention: the object of the present invention is to provide a kind of with low cost, anti-Potyvirus obvious results potato PVX, PVY, PLRV, PVS virus diagnose reagent process for making.The object of the present invention is achieved like this: sero-fast being prepared as follows of virus:
Implementation route
(1) phyto-indicator of Potyvirus is:
The main virus of table 1 potato is bred plant
| Virus | Numerous poisonous plant | Formal name used at school |
| Corium solani PLRV | The ocean wintercherry | Physalis floridana |
| Spend the bur datura in vain | Datura Stramonium | |
| Marmor upsilon PVY | The ocean wintercherry | Physalis floridana |
| Potato virus X PVX | Yellow seedling elm cigarette | Nicotiana tabaco |
| Potato virus S PVS | Yellow seedling elm cigarette | Nicotiana tabaco |
(2) gather in malicious source
Gather potato and produce the material that Tanaka has typical Potyvirus symptom, through the required drug source material of ELISA evaluation and screening, select the qualification result positive, light absorption value is higher, does auxiliary check with transmission electron microscope again, determines drug source material.
(3) separation, the breeding of the main virus of potato
The phyto-indicator that drug source material is inoculated in a series of health of chamber planting respectively carries out viral Separation Research, postvaccinal phyto-indicator must be every investigation in 2 days once, according to every group of phyto-indicator incidence, the virus that needs is isolated in screening, again through repeatedly inoculating other virus of elimination, purified virus, the virus inoculation with purifying expands numerous virus on numerous malicious material then, uses for purifying.
With the Potyvirus part low tempertaure storage of separation and purification, use for purifying as the viral malicious source propagative viruses of kit preparation in the future.
Virus is separated:
PVX: juice frictional inoculation, globe amaranth------the yellow seedling elm of finger tip green pepper---globe amaranth---cigarette of spending the bur datura in vain
PVY: juice frictional inoculation or the inoculation of aphid perishability, foreign wintercherry---yellow seedling elm cigarette
PVS: the inoculation of aphid perishability, globe amaranth---datura innoxia---Chenopodium amaranticolor---De Baini cigarette
PLRV: the inoculation of aphid persistence, foreign wintercherry---spend the bur datura in vain
(4) purification of the main virus of potato
Based on differential centrifugation,, adopt correlation method to purify according to the different virus feature.
1) several viral purification routes
PVX virus
↓
Host plant+2 times volumes (w/v) extract damping fluid and grind, and are centrifugal 3,000-4,000rpm, 20min
↓ ↓
Abandon the precipitation supernatant, centrifugal 10,000-12,000rpm, 20min
↓ ↓
Abandon supernatant precipitation+4%PEG damping fluid, stir 60min, centrifugal
10,000rpm，20min
↓ ↓
4 ℃ of static 60min of supernatant, centrifugal 10,000-12,000rpm, 20min abandons precipitation
↓ ↓
Abandon supernatant precipitation+4%PEG damping fluid, stir 120min, centrifugal
10,000-12,000rpm，20min
↓ ↓
Supernatant+extract spends the night for 4 ℃, and is centrifugal 10,000-12, and 000rpm, 20min abandons precipitation
↓ ↓
Supernatant 40,000rpm is centrifugal, and 90min abandons precipitation
↓ ↓
Abandoning supernatant precipitation+extraction damping fluid spends the night
↓
Pure virus
Pvy virus
↓
Host plant+2 times volumes (w/v) extract damping fluid, and three layers of filtered through gauze are centrifugal 7,800-8,000rpm, 20min
↓ ↓
Abandon in the precipitation supernatant and add 1%Triton X-100,4 ℃, it is centrifugal 7 that 120min vibrates, 800-8,000rpm, 20min
↓ ↓
Add the 4%PEG damping fluid in the supernatant, 4 ℃ of vibration 60min abandon precipitation
Foster 60min under the room temperature, centrifugal 7,800-8,000rpm, 20min
↓ ↓ precipitation suspends with the 0.02M phosphate buffer, adds 1%Triton X-100, abandons supernatant
4 ℃ are spent the night, centrifugal 5,100rpm, 10min
↓ ↓ supernatant 30% sucrose bed course is centrifugal 72,530rpm, and 150min abandons precipitation
↓ ↓ phosphate buffer the precipitation that suspends, 0.01MEDTA abandons 4 ℃ of chelating 240min of supernatant, and is centrifugal 5,100rpm, 10min
↓ ↓
Abandon precipitation supernatant and chlorination mixture of colours density gradient, centrifugal 128,000rpm, 180min
↓ ↓
Distribute and collect virus band adding equal-volume phosphate buffer (containing 0.01MEDTA)
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ of shaken overnight, centrifugal 5,500rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 160,400rpm, 60min
↓ ↓
Abandon supernatant phosphate buffer suspension precipitation, 4 ℃ are spent the night
↓
Pure virus
PLRV virus
↓
Host plant+2 times volumes (w/v) extract damping fluid liquid nitrogen grinding, shaken overnight under the room temperature
↓
Three layers of filtered through gauze, filtrate and chloroform (1: 1) emulsification 5 minutes, centrifugal 9,600-12,000rpm, 25min
↓ ↓
Abandon adding 8%PEG damping fluid in the precipitation supernatant, stir 120min under the room temperature
Centrifugal 9,600-12,000rpm, 45min
↓ ↓
In the precipitation+and the 0.01M phosphate buffer, 4 ℃ are spent the night, centrifugal 6,200rpm, 15min abandons supernatant
↓ ↓
It is centrifugal 11 to abandon the precipitation supernatant, 290rpm, 20min
↓ ↓
Supernatant sucrose bed course is centrifugal 72,530rpm, and 120min abandons precipitation
↓ ↓ phosphate buffer the precipitation that suspends, 4 ℃ are spent the night, centrifugal 7,840rpm, 10min abandons supernatant
↓ ↓
It is centrifugal 72 to abandon precipitation supernatant sucrose bed course, 530rpm, 120min
↓ ↓
Precipitation+phosphate buffer, 4 ℃ are spent the night, centrifugal 5,000rpm, 10min abandons supernatant
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 164,400rpm, 60min
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ of shaken overnight, centrifugal 5,500rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 160,400rpm, 60min
↓ ↓
Abandon supernatant phosphate buffer suspension precipitation, 4 ℃ are spent the night
↓
Pure virus
PVS virus
↓
Host plant+2 times volumes (w/v) extract damping fluid and grind, three layers of filtered through gauze,
Filtrate is centrifugal 5,000-6,000rpm, 20min
↓ ↓ supernatant+4%PEG and 1%Triton X-100, precipitation is abandoned in 4 ℃ of vibrations
60min is centrifugal 50,000rpm, 10min
↓ ↓ the abandon supernatant 0.05m phosphate buffer precipitation that suspends, centrifugal 10,000rpm, 20min
↓
Supernatant sucrose bed course is centrifugal 160,000rpm, 180min
↓ ↓
Abandon the supernatant 0.05m phosphate buffer precipitation that suspends, centrifugal 5,100rpm, 15min
↓ ↓
Sucrose density gradient, centrifugal 100,000rpm, 60min abandons precipitation
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 102,700rpm, 60min
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ are spent the night, centrifugal 5,100rpm, 15min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 128,500rpm, 40min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 102,700rpm, 60min
↓ ↓
Abandon supernatant precipitate phosphoric acid damping fluid and suspend, centrifugal 5,100rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 128,500rpm, 40min
↓
Pure virus
2) virus concentration is measured
Get each viral purification liquid and record each viral purity, concrete condition such as following table with ultraviolet spectrophotometer.
Ultraviolet spectrophotometer is surveyed virus quantity
| Virus | OD 260 | OD 280 | OD 260/OD 280 | Rna content % |
| PLRV | 8.6 | 10.62 | 0.81 | 3.2 |
| PVY | 2.8 | 2.33 | 1.20 | 5.4 |
| PVS | 2.93 | 2.31 | 1.27 | 5.3 |
| PVX | 2.97 | 2.50 | 1.19 | 5.9 |
(5) immunizing rabbit
After getting each sick degree dilution, use freund adjuvant and antigen 1: 1 mixing and emulsifying immunizing rabbit, injection is once injected 3 times altogether weekly, and injection volume is 2ml, 2ml, 3ml.After 3 weeks weekly ear neck arteries and veins get blood 20～30ml, survey antibody concentration, antibody concentration drops to top one half and carries out second time and inject.
1) injection examination
: fruend ' adguent+ physiological saline+1mg/ml antigen
2) extract serum
Place at the thick serum plastic centrifuge tube of the rabbit medium dip 30 degree angles of taking out, static spending the night under the room temperature (or 60～120 minutes, or ice bath 30 minutes), peel off and take out from the edge with bus moral suction pipe and separate out serum (centrifugal leucocyte-removing, red blood cell, the acquisition virus antiserum of removing), serum is deposited with several glass tubes.Once getting blood can get about viral antiserum 50ml.
(6) Immunoglobulin IgG preparation
1) Immunoglobulin IgG extracts
1ml serum adds 9ml water and adds 10ml water saturation ammonium sulfate, mixing, and room temperature left standstill 30～45 minutes, under 4 ℃ of conditions, 8000 revolutions per seconds, centrifugal 20 minutes, abandon supernatant, stay precipitation, with 1ml 0.5 * PBS damping fluid Hui Rong.Dialyse among 4 ℃ of 0.5 * PBS damping fluid, 500～1000ml, changed dislysate one time in 2 hours, change altogether 4 times.
2) viral globulin IgG concentration determination
Get 200 times of 280nm of globulin dilution and survey absorption value down
OD during virus globulin IgG1mg/ml
280=1.4
[IgG]＝OD
280×200/1.4
| PVX | PVY | PLRV | PVS | |
| OD280 | 0.125 | 0.113 | 0.130 | 0.107 |
| Concentration mg/ml | 17.85 | 16.14 | 18.57 | 15.28 |
(7) Immunoglobulin IgG enzyme labeling
1) enzyme labeled immunoassay globulin IgG-AP preparation
Label: alkaline phosphatase (AP)
Immunoglobulin IgG concentration: 1mg/ml.
1mg/ml IgG+24ml AP+2.6ul 25% glutaraldehyde solution mixing, room temperature was placed 2 hours, 4 ℃ of dialysis, the dialysis step is the same.
2) enzyme labeled immunoassay globulin IgG-AP preserves
Dialysis back liquid adds 5mg bovine serum albumin(BSA) (BSA), mixes the back and places 4 ℃ or-20 ℃ and preserve half a year～1 year.
(8) Immunoglobulin IgG and enzyme labeled immunoassay globulin IgG-AP quality determination
1) compare IgGOK=1 with known finished product Immunoglobulin IgG and enzyme labeled immunoassay globulin IgG-AP: 1000, IgG-APOK=1: 500
2) mensuration of the working concentration of immunoglobulin (Ig) and enzyme-labeled immunity globulin:
Enzyme-linked method is measured immunoglobulin (Ig) and enzyme-labeled immunity globulin working concentration, and IgG is cushioned liquid dilution 1/500,1/1000,1/1500,1/2000 with bag; IgG-AP dilutes 1/500,1/1000 with enzyme mark damping fluid, and the DAS-ELISA method is measured.Determine the relevant work concentration of IgG and IgG-AP.
Reagent of the present invention is applied to " toxicity-removing white potato potato seed quality supervision and test test center of the Ministry of Agriculture (Harbin) " 2003 and Ministry of Agriculture's field generaI investigation task in 2004, and the virus of some local agricultural technology centers and seed production unit detects.Therefore, utilize immunological technique to prepare Potyvirus antiserum detectable, have far-reaching social value and huge economic, simultaneously, for improving China's potato raw yield and quality monitoring system, carry out the virus supervision, improve China's potato yield and quality, win the international market, be significant.
Description of drawings: Fig. 1 is the sero-fast preparation technology's process flow diagram of the present invention's virus.
Embodiment: sero-fast being prepared as follows of virus: sero-fast being prepared as follows of virus:
Implementation route
(1) phyto-indicator of Potyvirus is:
The main virus of table 1 potato is bred plant
| Virus | Numerous poisonous plant | Formal name used at school |
| Corium solani PLRV | The ocean wintercherry | Physalis floridana |
| Spend the bur datura in vain | Datura Stramonium | |
| Marmor upsilon PVY | The ocean wintercherry | Physalis floridana |
| Potato virus X PVX | Yellow seedling elm cigarette | Nicotiana tabaco |
| Potato virus S PVS | Yellow seedling elm cigarette | Nicotiana tabaco |
(2) gather in malicious source
Gather potato and produce the material that Tanaka has typical Potyvirus symptom, through the required drug source material of ELISA evaluation and screening, select the qualification result positive, light absorption value is higher, does auxiliary check with transmission electron microscope again, determines drug source material.
(3) separation, the breeding of the main virus of potato
The phyto-indicator that drug source material is inoculated in a series of health of chamber planting respectively carries out viral Separation Research, postvaccinal phyto-indicator must be every investigation in 2 days once, according to every group of phyto-indicator incidence, the virus that needs is isolated in screening, again through repeatedly inoculating other virus of elimination, purified virus, the virus inoculation with purifying expands numerous virus on numerous malicious material then, uses for purifying.
With the Potyvirus part low tempertaure storage of separation and purification, use for purifying as the viral malicious source propagative viruses of kit preparation in the future.
Virus is separated:
PVX: juice frictional inoculation, globe amaranth------the yellow seedling elm of finger tip green pepper---globe amaranth---cigarette of spending the bur datura in vain.
PVY: juice frictional inoculation or the inoculation of aphid perishability, foreign wintercherry---yellow seedling elm cigarette.
PVS: the inoculation of aphid perishability, globe amaranth---datura innoxia---Chenopodium amaranticolor---De Baini cigarette.
PLRV: the inoculation of aphid persistence, foreign wintercherry---spend the bur datura in vain.
(4) purification of the main virus of potato
Based on differential centrifugation,, adopt correlation method to purify according to the different virus feature.
1) several viral purification routes
PVX virus
↓
Host plant+2 times volumes (w/v) extract damping fluid and grind, and are centrifugal 3,000-4,000rpm, 20min
↓ ↓
Abandon the precipitation supernatant, centrifugal 10,000-12,000rpm, 20min
↓ ↓
Abandon supernatant precipitation+4%PEG damping fluid, stir 60min, centrifugal
10,000rpm，20min
↓ ↓
4 ℃ of static 60min of supernatant, centrifugal 10,000-12,000rpm, 20min abandons precipitation
↓ ↓
Abandon supernatant precipitation+4%PEG damping fluid, stir 120min, centrifugal
10,000-12,000rpm，20min
↓ ↓
Supernatant+extract spends the night for 4 ℃, and is centrifugal 10,000-12, and 000rpm, 20min abandons precipitation
↓ ↓
Supernatant 40,000rpm is centrifugal, and 90min abandons precipitation
↓ ↓
Abandoning supernatant precipitation+extraction damping fluid spends the night
↓
Pure virus
Pvy virus
↓ host plant+2 times volumes (w/v) extract damping fluid, three layers of filtered through gauze, centrifugal 7,800-8,000rpm, 20min ↓ ↓ abandon and precipitate adding 1%Triton X-100 in the supernatant, 4 ℃, it is centrifugal 7 that 120min vibrates, 800-8,000rpm, 20min
↓ ↓
Add the 4%PEG damping fluid in the supernatant, 4 ℃ of vibration 60min abandon precipitation
Foster 60min under the room temperature, centrifugal 7,800-8,000rpm, 20min
↓ ↓ precipitation suspends with the 0.02M phosphate buffer, adds 1%Triton X-100, abandons supernatant
4 ℃ are spent the night, centrifugal 5,100rpm, 10min
↓ ↓ supernatant 30% sucrose bed course is centrifugal 72,530rpm, and 150min abandons precipitation
↓ ↓ phosphate buffer the precipitation that suspends, 0.01MEDTA abandons 4 ℃ of chelating 240min of supernatant, and is centrifugal 5,100rpm, 10min
↓ ↓
Abandon precipitation supernatant and chlorination mixture of colours density gradient, centrifugal 128,000rpm, 180min
↓ ↓
Distribute and collect virus band adding equal-volume phosphate buffer (containing 0.01MEDTA)
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ of shaken overnight, centrifugal 5,500rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 160,400rpm, 60min
↓ ↓
Abandon supernatant phosphate buffer suspension precipitation, 4 ℃ are spent the night
↓
Pure virus
PLRV virus
↓
Host plant+2 times volumes (w/v) extract damping fluid liquid nitrogen grinding, shaken overnight under the room temperature
↓
Three layers of filtered through gauze, filtrate and chloroform (1: 1) emulsification 5 minutes, centrifugal 9,600-12,000rpm, 25min
↓ ↓
Abandon adding 8%PEG damping fluid in the precipitation supernatant, stir 120min under the room temperature
Centrifugal 9,600-12,000rpm, 45min
↓ ↓
In the precipitation+and the 0.01M phosphate buffer, 4 ℃ are spent the night, centrifugal 6,200rpm, 15min abandons supernatant
↓ ↓
It is centrifugal 11 to abandon the precipitation supernatant, 290rpm, 20min
↓ ↓
Supernatant sucrose bed course is centrifugal 72,530rpm, and 120min abandons precipitation
↓ ↓ phosphate buffer the precipitation that suspends, 4 ℃ are spent the night, centrifugal 7,840rpm, 10min abandons supernatant
↓ ↓
It is centrifugal 72 to abandon precipitation supernatant sucrose bed course, 530rpm, 120min
↓ ↓
Precipitation+phosphate buffer, 4 ℃ are spent the night, centrifugal 5,000rpm, 10min abandons supernatant
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 164,400rpm, 60min
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ of shaken overnight, centrifugal 5,500rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 160,400rpm, 60min
↓ ↓ abandon supernatant phosphate buffer suspends and precipitates, and 4 ℃ are spent the night
↓
Pure virus
PVS virus
↓
Host plant+2 times volumes (w/v) extract damping fluid and grind, three layers of filtered through gauze,
Filtrate is centrifugal 5,000-6,000rpm, 20min
↓ ↓ supernatant+4%PEG and 1%Triton X-100, precipitation is abandoned in 4 ℃ of vibrations
60min is centrifugal 50,000rpm, and 10min ↓ ↓ abandon the supernatant 0.05m phosphate buffer precipitation that suspends, centrifugal 10,000rpm, 20min
↓
Supernatant sucrose bed course is centrifugal 160,000rpm, 180min
↓ ↓
Abandon the supernatant 0.05m phosphate buffer precipitation that suspends, centrifugal 5,100rpm, 15min
↓ ↓
Sucrose density gradient, centrifugal 100,000rpm, 60min abandons precipitation
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 102,700rpm, 60min
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ are spent the night, centrifugal 5,100rpm, 15min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 128,500rpm, 40min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 102,700rpm, 60min
↓ ↓
Abandon supernatant precipitate phosphoric acid damping fluid and suspend, centrifugal 5,100rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 128,500rpm, 40min
↓
Pure virus
2) virus concentration is measured
Get each viral purification liquid and record each viral purity, concrete condition such as following table with ultraviolet spectrophotometer.
Ultraviolet spectrophotometer is surveyed virus quantity
| Virus | OD 260 | OD 280 | OD 260/OD 280 | Rna content % |
| PLRV | 8.6 | 10.62 | 0.81 | 3.2 |
| PVY | 2.8 | 2.33 | 1.20 | 5.4 |
| PVS | 2.93 | 2.31 | 1.27 | 5.3 |
| PVX | 2.97 | 2.50 | 1.19 | 5.9 |
(5) immunizing rabbit
After getting each sick degree dilution, use freund adjuvant and antigen 1: 1 mixing and emulsifying immunizing rabbit, injection is once injected 3 times altogether weekly, and injection volume is 2ml, 2ml, 3ml.After 3 weeks weekly ear neck arteries and veins get blood 20～30ml, survey antibody concentration, antibody concentration drops to top one half and carries out second time and inject.
1) injection examination
: fruend ' adguent+ physiological saline+1mg/ml antigen
2) extract serum
Place at the thick serum plastic centrifuge tube of the rabbit medium dip 30 degree angles of taking out, static spending the night under the room temperature (or 60～120 minutes, or ice bath 30 minutes), peel off and take out from the edge with bus moral suction pipe and separate out serum (centrifugal leucocyte-removing, red blood cell, the acquisition virus antiserum of removing), serum is deposited with several glass tubes.Once getting blood can get about viral antiserum 50ml.
(6) Immunoglobulin IgG preparation
1) Immunoglobulin IgG extracts
1ml serum adds 9ml water and adds 10ml water saturation ammonium sulfate, mixing, and room temperature left standstill 30～45 minutes, under 4 ℃ of conditions, 8000 revolutions per seconds, centrifugal 20 minutes, abandon supernatant, stay precipitation, with 1ml 0.5 * PBS damping fluid Hui Rong.Dialyse among 4 ℃ of 0.5 * PBS damping fluid, 500～1000ml, changed dislysate one time in 2 hours, change altogether 4 times.
2) viral globulin IgG concentration determination
Get OD when surveying absorption value virus globulin IgG1mg/ml under 200 times of 280nm of globulin dilution
280=1.4.
[IgG]＝OD
280×200/1.4
| PVX | PVY | PLRV | PVS | |
| OD280 | 0.125 | 0.113 | 0.130 | 0.107 |
| Concentration mg/ml | 17.85 | 16.14 | 18.57 | 15.28 |
(7) Immunoglobulin IgG enzyme labeling
1) enzyme labeled immunoassay globulin IgG-AP preparation
Label: alkaline phosphatase (AP).
Immunoglobulin IgG concentration: 1mg/ml
1mg/ml IgG+24ml AP+2.6ul 25% glutaraldehyde solution mixing, room temperature was placed 2 hours, 4 ℃ of dialysis, the dialysis step is the same.
2) enzyme labeled immunoassay globulin IgG-AP preserves
Dialysis back liquid adds 5mg bovine serum albumin(BSA) (BSA), mixes the back and places 4 ℃ or-20 ℃ and preserve half a year～1 year.
(8) Immunoglobulin IgG and enzyme labeled immunoassay globulin IgG-AP quality determination
1) compare IgGOK=1 with known finished product Immunoglobulin IgG and enzyme labeled immunoassay globulin IgG-AP: 1000, IgG-APOK=1: 500
2) mensuration of the working concentration of immunoglobulin (Ig) and enzyme-labeled immunity globulin:
Enzyme-linked method is measured immunoglobulin (Ig) and enzyme-labeled immunity globulin working concentration, and IgG is cushioned liquid dilution 1/500,1/1000,1/1500,1/2000 with bag; IgG-AP dilutes 1/500,1/1000 with enzyme mark damping fluid, and the DAS-ELISA method is measured.Determine the relevant work concentration of IgG and IgG-AP.
Claims (2)
1. a potato PVX, PVY, PLRV, PVS virus diagnose reagent process for making, it is characterized in that: sero-fast being prepared as follows of virus: the first step is collected viral separatory phyto-indicator; Second step was gathered viral malicious source, identifying virus kind; Phyto-indicator separation of the 3rd step and purified virus; The 4th step phyto-indicator virus expands numerous; The purification of the main virus of the 5th step potato; The 6th step, viral purification liquid immunizing rabbit obtained antiserum; The extraction and the concentration determination of the 7th step antiserum immunoglobulin (Ig) (IgG); The enzyme labeling of the 8th step antiserum immune globulin; The Potyvirus detection sensitivity test of the 9th step; The working concentration of the tenth step immunoglobulin (Ig) and enzyme-labeled immunity globulin is measured;
Implementation route
(1) phyto-indicator of Potyvirus is:
The main virus of table 1 potato is bred plant
Virus Numerous poisonous plant Formal name used at school
Corium solani PLRV The ocean wintercherry Physalis floridana
Spend the bur datura in vain Datura Stramonium
Marmor upsilon PVY The ocean wintercherry Physalis floridana
Potato virus X PVX Yellow seedling elm cigarette Nicotiana tabaco
Potato virus S PVS Yellow seedling elm cigarette Nicotiana tabaco
(2) gather in malicious source
Gather potato and produce the material that Tanaka has typical Potyvirus symptom, through the required drug source material of ELISA evaluation and screening, select the qualification result positive, light absorption value is higher, does auxiliary check with transmission electron microscope again, determines drug source material.
(3) separation, the breeding of the main virus of potato
The phyto-indicator that drug source material is inoculated in a series of health of chamber planting respectively carries out viral Separation Research, postvaccinal phyto-indicator must be every investigation in 2 days once, according to every group of phyto-indicator incidence, the virus that needs is isolated in screening, again through repeatedly inoculating other virus of elimination, purified virus, the virus inoculation with purifying expands numerous virus on numerous malicious material then, uses for purifying.
With the Potyvirus part low tempertaure storage of separation and purification, use for purifying as the viral malicious source propagative viruses of kit preparation in the future.
Virus is separated:
PVX: juice frictional inoculation, globe amaranth------the yellow seedling elm of finger tip green pepper---globe amaranth---cigarette of spending the bur datura in vain
PVY: juice frictional inoculation or the inoculation of aphid perishability, foreign wintercherry---yellow seedling elm cigarette
PVS: the inoculation of aphid perishability, globe amaranth---datura innoxia---Chenopodium amaranticolor---De Baini cigarette
PLRV: the inoculation of aphid persistence, foreign wintercherry---spend the bur datura in vain
(4) purification of the main virus of potato
Based on differential centrifugation,, adopt correlation method to purify according to the different virus feature.
1) several viral purification routes
PVX virus
↓
Host plant+2 times volumes (w/v) extract damping fluid and grind, and are centrifugal 3,000-4,000rpm, 20min
↓ ↓
Abandon the precipitation supernatant, centrifugal 10,000-12,000rpm, 20min
↓ ↓
Abandon supernatant precipitation+4%PEG damping fluid, stir 60min, centrifugal
10,000rpm，20min
↓ ↓
4 ℃ of static 60min of supernatant, centrifugal 10,000-12,000rpm, 20min abandons precipitation
↓ ↓
Abandon supernatant precipitation+4%PEG damping fluid, stir 120min, centrifugal
10,000-12,000rpm，20min
↓ ↓
Supernatant+extract spends the night for 4 ℃, and is centrifugal 10,000-12, and 000rpm, 20min abandons precipitation
↓ ↓
Supernatant 40,000rpm is centrifugal, and 90min abandons precipitation
↓ ↓
Abandoning supernatant precipitation+extraction damping fluid spends the night
↓
Pure virus
Pvy virus
↓ host plant+2 times volumes (w/v) extract damping fluid, three layers of filtered through gauze, centrifugal 7,800-8,000rpm, 20min ↓ ↓ abandon and precipitate adding 1%Triton X-100 in the supernatant, 4 ℃, it is centrifugal 7 that 120min vibrates, 800-8,000rpm, 20min
↓ ↓
Add the 4%PEG damping fluid in the supernatant, 4 ℃ of vibration 60min abandon precipitation
Foster 60min under the room temperature, centrifugal 7,800-8,000rpm, 20min
↓ ↓ precipitation suspends with the 0.02M phosphate buffer, adds 1%Triton X-100, abandons supernatant
4 ℃ are spent the night, centrifugal 5,100rpm, 10min
↓ ↓ supernatant 30% sucrose bed course is centrifugal 72,530rpm, and 150min abandons precipitation
↓ ↓ phosphate buffer the precipitation that suspends, 0.01MEDTA abandons 4 ℃ of chelating 240min of supernatant, and is centrifugal 5,100rpm, 10min
↓ ↓
Abandon precipitation supernatant and chlorination mixture of colours density gradient, centrifugal 128,000rpm, 180min
↓ ↓
Distribute and collect virus band adding equal-volume phosphate buffer (containing 0.01MEDTA)
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ of shaken overnight, centrifugal 5,500rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 160,400rpm, 60min
↓ ↓
Abandon supernatant phosphate buffer suspension precipitation, 4 ℃ are spent the night
↓
Pure virus
PLRV virus
↓
Host plant+2 times volumes (w/v) extract damping fluid liquid nitrogen grinding, shaken overnight under the room temperature
↓
Three layers of filtered through gauze, filtrate and chloroform (1: 1) emulsification 5 minutes, centrifugal 9,600-12,000rpm, 25min
↓ ↓
Abandon adding 8%PEG damping fluid in the precipitation supernatant, stir 120min under the room temperature
Centrifugal 9,600-12,000rpm, 45min
↓ ↓
In the precipitation+and the 0.01M phosphate buffer, 4 ℃ are spent the night, centrifugal 6,200rpm, 15min abandons supernatant
↓ ↓
It is centrifugal 11 to abandon the precipitation supernatant, 290rpm, 20min
↓ ↓
Supernatant sucrose bed course is centrifugal 72,530rpm, and 120min abandons precipitation
↓ ↓ phosphate buffer the precipitation that suspends, 4 ℃ are spent the night, centrifugal 7,840rpm, 10min abandons supernatant
↓ ↓
It is centrifugal 72 to abandon precipitation supernatant sucrose bed course, 530rpm, 120min
↓ ↓
Precipitation+phosphate buffer, 4 ℃ are spent the night, centrifugal 5,000rpm, 10min abandons supernatant
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 164,400rpm, 60min
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ of shaken overnight, centrifugal 5,500rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 114,500rpm, 45min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 160,400rpm, 60min
↓ ↓
Abandon supernatant phosphate buffer suspension precipitation, 4 ℃ are spent the night
↓
Pure virus
PVS virus
↓
Host plant+2 times volumes (w/v) extract damping fluid and grind, three layers of filtered through gauze,
Filtrate is centrifugal 5,000-6,000rpm, 20min
↓ ↓ supernatant+4%PEG and 1%Triton X-100, precipitation is abandoned in 4 ℃ of vibrations
60min is centrifugal 50,000rpm, and 10min ↓ ↓ abandon the supernatant 0.05m phosphate buffer precipitation that suspends, centrifugal 10,000rpm, 20min
↓
Supernatant sucrose bed course is centrifugal 160,000rpm, 180min
↓ ↓
Abandon the supernatant 0.05m phosphate buffer precipitation that suspends, centrifugal 5,100rpm, 15min
↓ ↓
Sucrose density gradient, centrifugal 100,000rpm, 60min abandons precipitation
↓ distribute to collect the virus band, the phosphate buffer dilution, centrifugal 102,700rpm, 60min
↓ ↓
Abandon the supernatant phosphate buffer precipitation that suspends, 4 ℃ are spent the night, centrifugal 5,100rpm, 15min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 128,500rpm, 40min
↓
Distribute to collect the virus band, the phosphate buffer dilution, centrifugal 102,700rpm, 60min
↓ ↓
Abandon supernatant precipitate phosphoric acid damping fluid and suspend, centrifugal 5,100rpm, 10min
↓ ↓
Abandon the precipitation sucrose density gradient, centrifugal 128,500rpm, 40min
↓
Pure virus
2) virus concentration is measured
Get each viral purification liquid and record each viral purity, concrete condition such as following table with ultraviolet spectrophotometer.
Ultraviolet spectrophotometer is surveyed virus quantity
Virus OD
260 OD
280 OD
260/OD
280 Rna content %
PLRV 8.6 10.62 0.81 3.2
PVY 2.8 2.33 1.20 5.4
PVS 2.93 2.31 1.27 5.3
PVX 2.97 2.50 1.19 5.9
(5) immunizing rabbit
After getting each sick degree dilution, use freund adjuvant and antigen 1: 1 mixing and emulsifying immunizing rabbit, injection is once injected 3 times altogether weekly, and injection volume is 2ml, 2ml, 3ml.After 3 weeks weekly ear neck arteries and veins get blood 20～30ml, survey antibody concentration, antibody concentration drops to top one half and carries out second time and inject.
1) injection examination
: fruend ' adguent+ physiological saline+1mg/ml antigen
2) extract serum
Place at the thick serum plastic centrifuge tube of the rabbit medium dip 30 degree angles of taking out, static spending the night under the room temperature (or 60～120 minutes, or ice bath 30 minutes), peel off and take out from the edge with bus moral suction pipe and separate out serum (centrifugal leucocyte-removing, red blood cell, the acquisition virus antiserum of removing), serum is deposited with several glass tubes.Once getting blood can get about viral antiserum 50ml.
(6) Immunoglobulin IgG preparation
1) Immunoglobulin IgG extracts
1ml serum adds 9ml water and adds 10ml water saturation ammonium sulfate, mixing, and room temperature left standstill 30～45 minutes, under 4 ℃ of conditions, 8000 revolutions per seconds, centrifugal 20 minutes, abandon supernatant, stay precipitation, with 1ml0.5 * PBS damping fluid Hui Rong.Dialyse among 4 ℃ of 0.5 * PBS damping fluid, 500～1000ml, changed dislysate one time in 2 hours, change altogether 4 times.
2) viral globulin IgG concentration determination
Get 200 times of 280nm of globulin dilution and survey absorption value down
OD during virus globulin IgG1mg/ml
280=1.4
[IgG]＝OD
280×200/1.4
PVX PVY PLRV PVS
OD280 0.125 0.113 0.130 0.107
Concentration mg/ml 17.85 16.14 18.57 15.28
(7) Immunoglobulin IgG enzyme labeling
1) enzyme labeled immunoassay globulin IgG-AP preparation
Label: alkaline phosphatase (AP)
Immunoglobulin IgG concentration: 1mg/ml
1mg/ml IgG+24ml AP+2.6ul 25% glutaraldehyde solution
Mixing, room temperature was placed 2 hours, 4 ℃ of dialysis, the dialysis step is the same
2) enzyme labeled immunoassay globulin IgG-AP preserves
Dialysis back liquid adds 5mg bovine serum albumin(BSA) (BSA), mixes the back and places
4 ℃ or-20 ℃ are preserved half a year～1 year
(8) Immunoglobulin IgG and enzyme labeled immunoassay globulin IgG-AP quality determination
1) compare IgGOK=1 with known finished product Immunoglobulin IgG and enzyme labeled immunoassay globulin IgG-AP: 1000, IgG-APOK=1: 500
2) mensuration of the working concentration of immunoglobulin (Ig) and enzyme-labeled immunity globulin:
Enzyme-linked method is measured immunoglobulin (Ig) and enzyme-labeled immunity globulin working concentration, and IgG is cushioned liquid dilution 1/500,1/1000,1/1500,1/2000 with bag; IgG-AP dilutes 1/500,1/1000 with enzyme mark damping fluid, and the DAS-ELISA method is measured.Determine the relevant work concentration of IgG and IgG-AP.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212130A (en) * | 2010-04-07 | 2011-10-12 | 上海文渊阁生物科技有限公司 | Separation and purification preparation method for high-flux antiserum |
| CN102854313A (en) * | 2012-09-29 | 2013-01-02 | 黑龙江省农业科学院植物脱毒苗木研究所 | Preparation method of potato virus Y positive reference substance |
| CN106702023A (en) * | 2016-12-30 | 2017-05-24 | 中国烟草总公司广东省公司 | Rapid identification method of PVX viruses and application thereof |
| CN109136323A (en) * | 2018-09-21 | 2019-01-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of fixed diagnostic electronmicroscopy method of separation in situ of marmor upsilon plastochondria |
-
2004
- 2004-12-17 CN CN 200410044129 patent/CN1869698A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212130A (en) * | 2010-04-07 | 2011-10-12 | 上海文渊阁生物科技有限公司 | Separation and purification preparation method for high-flux antiserum |
| CN102854313A (en) * | 2012-09-29 | 2013-01-02 | 黑龙江省农业科学院植物脱毒苗木研究所 | Preparation method of potato virus Y positive reference substance |
| CN106702023A (en) * | 2016-12-30 | 2017-05-24 | 中国烟草总公司广东省公司 | Rapid identification method of PVX viruses and application thereof |
| CN109136323A (en) * | 2018-09-21 | 2019-01-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of fixed diagnostic electronmicroscopy method of separation in situ of marmor upsilon plastochondria |
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