CN102854313A - Preparation method of potato virus Y positive reference substance - Google Patents
Preparation method of potato virus Y positive reference substance Download PDFInfo
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- CN102854313A CN102854313A CN2012103715971A CN201210371597A CN102854313A CN 102854313 A CN102854313 A CN 102854313A CN 2012103715971 A CN2012103715971 A CN 2012103715971A CN 201210371597 A CN201210371597 A CN 201210371597A CN 102854313 A CN102854313 A CN 102854313A
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Abstract
The invention relates to a preparation method of a potato virus Y (PVY) positive reference substance, which relates to a preparation method of a virus positive reference substance. The problems in the existing PVY virus detection kit that the positive reference is expensive in price, short in quality guarantee period and unfavorable for being popularized can be solved. The preparation method comprises following steps of A, grinding PVY breeding host leaves and extracted buffering liquid after being mixed, and filtering to obtain virus juice; B, mixing and heating gelatin and the extracted buffering liquid until being molten, adding cane sugar, pouring the molten solution into the virus juice to be uniformly mixed, and pouring the mixed solution into a penicillin bottle to be dried in a freezing way to obtain the PVY frozen powder. The prepared PVY positive reference substance is low in price, and the price is one third of the imported PVY positive reference substance; the quality guarantee period can reach more than 3 years, the detection result value of double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) reaches more than 2.000; and being matched with the PVY virus detection kit produced by Heilongjiang Province Academy of Agriculture to use, the PVY positive reference substance is favorable for being popularized to use.
Description
Technical field
The present invention relates to the preparation method of virus-positive reference substance.
Background technology
Potato is Chinese the fourth-largest cereal crops, yet the integral production level but is lower than world average level, and wherein most important reason is exactly the yield and quality that virosis has had a strong impact on potato.
Marmor upsilon (Potato virus Y, PVY) be one of the important virus of harm potato, be distributed in each potato producing region, the world, can propagate in non-persistent mode by aphid, cause potato degeneration, yield reducation, the underproduction can reach more than 80% when serious.Particularly synergism often occurs with potato virus X (PVX) in PVY on solanaceous crops, has aggravated the harm of virosis, causes huge economic loss.
At present effectively preventing measure is to promote virus-free seed potato and strengthen testing, effectively detection method is the important measures that guarantee production safety, conventional sense generally adopts virus detection kit both at home and abroad, use the DAS-ELISA technology and carry out the PVY detection, laboratory test results need to be whether the marmor upsilon freeze-dried powder comes judged result accurate with the positive control of virus, can carry out smoothly the vital effect that plays to experiment, and this kit whole world only has ten enterprise marketings of less than, present domestic PVY virus detects almost completely dependence on import kit, expensive, limit popularizing of detection technique, thereby limited the raising of seed potato quality and the development of industry.In addition, there is following shortcoming in the import reagent box: (1) is expensive: the positive control in the import reagent box is expensive, about 300 yuan of every bottle of needs; (2) shelf-life is short: the positive control shelf-life in the import reagent box is about 1 year, dilute or dilute rear multigelation such as the freeze-dried powder to positive control, shelf-life also will shorten greatly, and the testing result value is very low, is unfavorable for the judgement of testing result; (3) be unfavorable for popularizing: since expensive, only have 1 bottle of positive control in the 1 cover marmor upsilon kit, owing to need repeated detection, each detection all needs to use positive control, therefore, and through multigelation, shelf-life shortens, and detected value is low, affects testing result.Thereby the application of the detection kit of marmor upsilon is restricted, is unfavorable for popularizing.
Summary of the invention
The present invention seeks in order to solve the problem that positive control in the existing PVY virus detection kit is expensive, the shelf-life short and be unfavorable for popularizing, and the preparation method of marmor upsilon positive reference substance is provided.
The preparation method of marmor upsilon positive reference substance realizes according to the following steps:
One, grinds after the Extraction buffer of the PVY host egg blade of 10g and 120mL is mixed, then filter with double gauze, obtain viral juice;
Two, the gelatin of 0.06～0.12g and the Extraction buffer of 80mL are mixed, be heated to thawing, then the sucrose that adds 6～10g, continue to be heated to sucrose and melt, pour mixing in the viral juice into, pour into again in the penicillin bottle, every bottle adds 1～2ml, cover bottle stopper and put into freeze drier and drain, obtain the freeze-dried powder of marmor upsilon, namely finish the preparation of marmor upsilon positive reference substance;
Wherein in the step 1 the every 100mL of Extraction buffer by the NaCL of PVP-40000, the 8.0g of 20g, the KH of 0.2g
2PO
4, 2.9g Na
2HPO
412H
2The KCL of O, 0.2g, the NaN of 0.2g
3, the Tween20 of 0.5mL and surplus distilled water form.
The beneficial effect of the marmor upsilon positive reference substance of the present invention's preparation is: (1) price is low: every bottle of price of marmor upsilon positive control is 100 yuan, and the price of import positive control is about 300 yuan, is 1/3 of import price;
(2) long shelf-life, detection sensitivity is high: product has been carried out the tracking test of shelf-life, detection sensitivity, and this shelf life of products of empirical tests reached more than 3 years, and DAS-ELISA testing result value reaches more than 2.000;
(3) be beneficial to and popularize: cheap, testing result accurately, long shelf-life, the PVY virus detection kit that cooperates Heilongjiang Institute of Agricultural Sciences to produce uses, and is beneficial to popularize.
Embodiment
Embodiment one: the preparation method of present embodiment marmor upsilon positive reference substance realizes according to the following steps:
One, grinds after the Extraction buffer of the PVY host egg blade of 10g and 120mL is mixed, then filter with double gauze, obtain viral juice;
Two, the gelatin of 0.06～0.12g and the Extraction buffer of 80mL are mixed, be heated to thawing, then the sucrose that adds 6～10g, continue to be heated to sucrose and melt, pour mixing in the viral juice into, pour into again in the penicillin bottle, every bottle adds 1～2ml, cover bottle stopper and put into freeze drier and drain, obtain the freeze-dried powder of marmor upsilon, namely finish the preparation of marmor upsilon positive reference substance;
Wherein in the step 1 the every 100mL of Extraction buffer by the NaCL of PVP-40000, the 8.0g of 20g, the KH of 0.2g
2PO
4, 2.9g Na
2HPO
412H
2The KCL of O, 0.2g, the NaN of 0.2g
3, the Tween20 of 0.5mL and surplus distilled water form.
The acquisition of PVY host egg blade in the present embodiment: 1. in the potato raw long season, the field gathers the potato leaf that floral leaf or downright bad symptom are arranged, take back the laboratory, detect as the blade of PVY virus is stored in-20 ℃ of refrigerators through serology DAS-ELISA method, as PVY poison source; 2. the mode by frictional inoculation is inoculated into PVY poison source on the host egg " yellow seedling elm cigarette " of PVY, the docking plant from the inoculation from every 10 days, detect with serology DAS-ELISA method, gather when the testing result value reaches more than 2.000, carry out the preparation of marmor upsilon positive reference substance.
Put into freeze drier in the present embodiment and drain, the technological process of freeze-dried powder that obtains marmor upsilon is as follows:
1. freeze drier is opened, temperature transfers to-50 ℃;
2. when temperature reaches-50 ℃, penicillin bottle is put into freeze drier drained 3 hours;
3.-40 ℃, drained 3 hours;
4.-30 ℃, drained 3 hours;
5.-20 ℃, drained 3 hours;
6.-10 ℃, drained 3 hours;
7.0 ℃, drained 3 hours;
8.10 ℃, drained 1 hour;
9.20 ℃, drained 1 hour;
10.30 ℃, drained 1 hour;
11. vacuum state gland;
12. taking-up penicillin bottle.
The PVY virus detection kit that the marmor upsilon positive reference substance for preparing in the present embodiment cooperates Heilongjiang Institute of Agricultural Sciences to produce uses; The principal ingredient of the PVY virus detection kit that Heilongjiang Institute of Agricultural Sciences produces is: antibody (IgG), enzyme labelled antibody (IgG-Ap), sample extraction damping fluid, lavation buffer solution, coated damping fluid, enzyme mark damping fluid, substrate buffer solution, substrate, marmor upsilon positive control and negative control.
Embodiment two: what present embodiment and embodiment one were different is in the step 2 gelatin of 0.1g and the Extraction buffer of 80mL to be mixed, and is heated to thawing, then adds the sucrose of 8g.Other step and parameter are identical with embodiment one.
Embodiment:
The preparation method of marmor upsilon positive reference substance realizes according to the following steps:
One, grinds after the Extraction buffer of the PVY host egg blade of 10g and 120mL is mixed, then filter with double gauze, obtain viral juice;
Two, the gelatin of 0.1g and the Extraction buffer of 80mL are mixed, be heated to thawing, then the sucrose that adds 8g, continue to be heated to sucrose and melt, pour mixing in the viral juice into, pour into again in the penicillin bottle, every bottle adds 1～2ml, cover bottle stopper and put into freeze drier and drain, obtain the freeze-dried powder of marmor upsilon, namely finish the preparation of marmor upsilon positive reference substance;
Wherein in the step 1 the every 100mL of Extraction buffer by the NaCL of PVP-40000, the 8.0g of 20g, the KH of 0.2g
2PO
4, 2.9g Na
2HPO
412H
2The KCL of O, 0.2g, the NaN of 0.2g
3, the Tween20 of 0.5mL and surplus distilled water form.
Put into freeze drier in the present embodiment and drain, the technological process of freeze-dried powder that obtains marmor upsilon is as follows:
1. freeze drier is opened, temperature transfers to-50 ℃;
2. when temperature reaches-50 ℃, penicillin bottle is put into freeze drier drained 3 hours;
3.-40 ℃, drained 3 hours;
4.-30 ℃, drained 3 hours;
5.-20 ℃, drained 3 hours;
6.-10 ℃, drained 3 hours;
7.0 ℃, drained 3 hours;
8.10 ℃, drained 1 hour;
9.20 ℃, drained 1 hour;
10.30 ℃, drained 1 hour;
11. vacuum state gland;
12. taking-up penicillin bottle.
The PVY virus detection kit that the marmor upsilon positive reference substance for preparing in the present embodiment cooperates Heilongjiang Institute of Agricultural Sciences to produce uses, the tracking test that carries out sensitivity, and DAS-ELISA testing result value reaches more than 2.000.
Claims (2)
1. the preparation method of marmor upsilon positive reference substance is characterized in that the preparation method of marmor upsilon positive reference substance realizes according to the following steps:
One, grinds after the Extraction buffer of the PVY host egg blade of 10g and 120mL is mixed, then filter with double gauze, obtain viral juice;
Two, the gelatin of 0.06～0.12g and the Extraction buffer of 80mL are mixed, be heated to thawing, then the sucrose that adds 6～10g, continue to be heated to sucrose and melt, pour mixing in the viral juice into, pour into again in the penicillin bottle, every bottle adds 1～2ml, cover bottle stopper and put into freeze drier and drain, obtain the freeze-dried powder of marmor upsilon, namely finish the preparation of marmor upsilon positive reference substance;
Wherein in the step 1 the every 100mL of Extraction buffer by the NaCL of PVP-40000, the 8.0g of 20g, the KH of 0.2g
2PO
4, 2.9g Na
2HPO
412H
2The KCL of O, 0.2g, the NaN of 0.2g
3, the Tween20 of 0.5mL and surplus distilled water form.
2. the preparation method of marmor upsilon positive reference substance according to claim 1 is characterized in that in the step 2 gelatin of 0.1g and the Extraction buffer of 80mL being mixed, and is heated to thawing, then adds the sucrose of 8g.
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| CN2012103715971A CN102854313A (en) | 2012-09-29 | 2012-09-29 | Preparation method of potato virus Y positive reference substance |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114034862A (en) * | 2021-11-29 | 2022-02-11 | 内蒙古中加农业生物科技有限公司 | Potato seed virus detection method |
Citations (5)
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| JPS62178523A (en) * | 1986-01-23 | 1987-08-05 | ベ−リングヴエルケ・アクチエンゲゼルシヤフト | Viral antigen medicine and manufacture |
| CN1869698A (en) * | 2004-12-17 | 2006-11-29 | 黑龙江省农业科学院植物脱毒苗木研究所 | Preparation technical method of potato PVX, PVY< PLRV, PVS virus diagnostic reagent |
| CN101287449A (en) * | 2002-04-11 | 2008-10-15 | 米迪缪尼疫苗股份有限公司 | Preservation of bioactive materials by spray drying |
| CN101312742A (en) * | 2005-09-16 | 2008-11-26 | 梅瑞尔有限公司 | Stabilizers for freeze-dried vaccines |
| CN101633909A (en) * | 2009-08-13 | 2010-01-27 | 武华 | Attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome |
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2012
- 2012-09-29 CN CN2012103715971A patent/CN102854313A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62178523A (en) * | 1986-01-23 | 1987-08-05 | ベ−リングヴエルケ・アクチエンゲゼルシヤフト | Viral antigen medicine and manufacture |
| CN101287449A (en) * | 2002-04-11 | 2008-10-15 | 米迪缪尼疫苗股份有限公司 | Preservation of bioactive materials by spray drying |
| CN1869698A (en) * | 2004-12-17 | 2006-11-29 | 黑龙江省农业科学院植物脱毒苗木研究所 | Preparation technical method of potato PVX, PVY< PLRV, PVS virus diagnostic reagent |
| CN101312742A (en) * | 2005-09-16 | 2008-11-26 | 梅瑞尔有限公司 | Stabilizers for freeze-dried vaccines |
| CN101633909A (en) * | 2009-08-13 | 2010-01-27 | 武华 | Attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome |
Non-Patent Citations (1)
| Title |
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| 蒋国司 等: "马铃薯病毒病感染率之群体测试", 《植物病理学会刊》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114034862A (en) * | 2021-11-29 | 2022-02-11 | 内蒙古中加农业生物科技有限公司 | Potato seed virus detection method |
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Application publication date: 20130102 |