CN103924005A - Detection method for influenza virus inactivation test verification - Google Patents
Detection method for influenza virus inactivation test verification Download PDFInfo
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- CN103924005A CN103924005A CN201310016065.0A CN201310016065A CN103924005A CN 103924005 A CN103924005 A CN 103924005A CN 201310016065 A CN201310016065 A CN 201310016065A CN 103924005 A CN103924005 A CN 103924005A
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Abstract
The invention relates to a detection method for influenza virus inactivation test verification, and the detection method is characterized in that Vero cell method is employed for detecting influenza virus inactivation verification, a to-be detected sample is subjected to original-time dilution, 10<-1> time dilution and 10<-2> time dilution, and the processed sample with each same dilutability is used to inoculate six bottles of cells. Each bottle of cells is inoculated with 1 mL of the processed sample, adsorption is carried out at 33 DEG C for 60 min, then the culture solution is poured out and 5 mL of a virus culture solution is added. Culturing is carried out at 33 DEG C for 72 h, so that culture solutions are harvested and used for blood coagulation. The harvest solutions with one same dilutability are mixed according to the ratio of 1:1, and blind passage is performed for two generations according to the above method, harvest samples are used for blood coagulation, if a third-generation blood coagulation result is negative, the influenza virus inactivation test verification is qualified, otherwise, the influenza virus inactivation test verification is unqualified. The method helps to solve the chick embryo source problem once avian influenza breaks out, and has the advantages of simple process, high accuracy, good flexibility and the like by employing Vero cells as a culture medium.
Description
Technical field
The present invention relates to a kind of detection method of biotechnological formulation, particularly relate to a kind of detection method of inactivated influenza virus verification experimental verification, belong to biological technical field.
Background technology
What the detection method of traditional inactivated influenza virus verification experimental verification was applied is chick embryo method.Allantoic fluid sample after inactivation of virus is done to 10 times of serial dilutions, get former times, 10
-1and 10
-2the doubly virus liquid of dilution inoculated into chick embryo allantoic cavity respectively, 10 pieces of instar chicken embryos on the 9th～11 of every group of inoculation, every embryonic breeding kind 0.2ml, puts 33～35 DEG C and cultivates 72 hours.Dead not counting in 24 hours, every group of chicken embryo must at least survive 80%, in the chicken embryo of survival, every embryo is got 0.5ml allantoic fluid, after mixing by group, then a blind passage generation, 10 pieces of embryos of every group of each inoculation, every embryonic breeding kind 0.2ml,, after 72 hours, getting allantoic fluid and carry out hemagglutination test through 33～35 DEG C of cultivations, should not there is not hemagglutination in result.
Existing " checking of influenza inactivation test " is " three of pharmacopeia " method for chicken embryo influenza vaccines " inactivation test checking ", be chicken embryo blind passage 2 generation blood clotting result negative be judged to qualified.This method is applicable to inspection chicken embryo influenza vaccines.Influenza virus after the adaptation in tens generations, strengthens the cell adapted ability of Vero on Vero cell, chicken embryo adaptive faculty is weakened relatively to Reduced susceptibility.Whether deactivation is thorough to check virus with chicken embryo as host again, and the accuracy of result needs to be proved.Once Avian Influenza, the source of chicken embryo is a problem especially.
Summary of the invention
The object of the invention is to solve the problems referred to above that prior art exists, disclose a kind of method of the Vero of employing cell detection inactivated influenza virus verification experimental verification.The method of this new inactivated influenza virus checking that the present invention provides adopts Vero cell as viral culture medium, by the investigation to sensitivity, and simultaneous test, prove that it is feasible doing influenza virus " inactivation test checking " method with Vero cell.
The technical scheme that the present invention provides is: the detection method of this inactivated influenza virus verification experimental verification, be characterized in applying Vero cell method and detect the deactivation checking of influenza virus, and there are following steps:
(1) H3N2 type influenza virus (lot number is 20090731-5) is diluted to every hole and goes out 1-5 spot, be diluted to 10
-7, 0.5 × 10
-7, with this extent of dilution inoculated into chick embryo, square vase, 6 orifice plates respectively:
Chicken embryo: 10
-7, 0.5 × 10
-7each 6 pieces of embryos, 0.2ml/ embryo;
Square vase: 10
-7, 0.5 × 10
-7each 6 holes, 0.5ml/ bottle;
6 orifice plates: 10
-7, 0.5 × 10
-7each 6 holes, 0.5ml/ hole;
Chicken embryo passed for 2 generations one to one, and detected respectively blood clotting titre, result of determination; Square vase passed for 2 generations one to one, and detected respectively blood clotting titre, result of determination; 6 porocyte culture plates pass 1 generation, plaque test, result of determination;
(2) utilize Veo cell method to detect inactivated influenza virus verification experimental verification:
T25 Tissue Culture Flask Cultivation of Vero, for subsequent use after growing up to individual layer;
By extremely former times, 10 of diluted sample to be checked
-1, 10
-2, 6 bottles of cells of each extent of dilution inoculation;
Every bottle of cell inoculation sample 1ml, outwells after 33 DEG C of absorption 60min, adds virus-culturing fluid 5ml;
33 DEG C of cultivations are gathered in the crops respectively nutrient solution after 72 hours and are done blood clotting;
Same extent of dilution harvest liquid was mixed by 1: 1, and in 2 generations of blind passage more as stated above, results sample does blood clotting;
Result: third generation blood clotting result is negative be judged to qualified, otherwise defective.
Compared with prior art, the invention has the beneficial effects as follows: the detection method (cell method) that a kind of inactivated influenza virus checking is provided, adopt Vero cell as viral culture medium, by investigation and the simultaneous test of sensitivity, proving to set up Vero cell method is feasible as inactivated influenza virus verification experimental verification method.By extremely former times, 10 of diluted sample to be checked
-1, 10
-2, 6 bottles of cells of each extent of dilution inoculation.Every bottle of cell inoculation sample 1ml, outwells after 33 DEG C of absorption 60min, adds virus-culturing fluid 5ml.33 DEG C of cultivations are gathered in the crops respectively nutrient solution after 72 hours and are done blood clotting.Same extent of dilution harvest liquid was mixed to 2 generations of blind passage more as stated above by 1: 1.Results sample does blood clotting.Third generation blood clotting result is negative be judged to qualified, otherwise defective.Once this test method has solved Avian Influenza, chicken embryo carry out source problem.Adopt Vero cell as culture medium, there is the advantages such as method is easy, accuracy is high, handiness is good.
Embodiment
Embodiment 1
H3N2 type influenza virus (lot number is 20090731-5) is diluted to every hole and goes out 1-5 spot, be diluted to 10
-7, 0.5 × 10
-7.With this extent of dilution inoculated into chick embryo, square vase, 6 orifice plates respectively.Chicken embryo: 10
-7, 0.5 × 10
-7each 6 pieces of embryos, 0.2ml/ embryo; Square vase: 10
-7, 0.5 × 10
-7each 6 holes, 0.5ml/ bottle; 6 orifice plates: 10
-7, 0.5 × 10
-7each 6 holes, 0.5ml/ hole.Chicken embryo passed for 2 generations one to one, and detected respectively blood clotting titre, result of determination.Square vase passed for 2 generations one to one, and detected respectively blood clotting titre, result of determination.6 porocyte culture plates pass 1 generation, plaque test, result of determination.
The results are shown in following table:
Remarks: 1, "+" represents that positive, "-" represents feminine gender.
2, generation result there will be false positive, therefore select two generation result of determination.
When viral dilution to 0.5 × 10
-7time, each method positive detects per-cent and is: square vase 100%, chicken embryo 50%, 6 orifice plates 67%.Detect inactivated influenza virus assay sensitivity with Vero cell square vase the highest.
Embodiment 2
Utilize Veo cell method to detect inactivated influenza virus verification experimental verification: T25 Tissue Culture Flask Cultivation of Vero, for subsequent use after growing up to individual layer.By extremely former times, 10 of diluted sample to be checked
-1, 10
-2, 6 bottles of cells of each extent of dilution inoculation.Every bottle of cell inoculation sample 1ml, outwells after 33 DEG C of absorption 60min, adds virus-culturing fluid 5ml.33 DEG C of cultivations are gathered in the crops respectively nutrient solution after 72 hours and are done blood clotting.Same extent of dilution harvest liquid was mixed to 2 generations of blind passage more as stated above by 1: 1.Results sample does blood clotting.Result: third generation blood clotting result is negative be judged to qualified, otherwise defective.(cell method)
Allantoic fluid sample after inactivation of virus is done to 10 times of serial dilutions, get former times, 10
-1and 10
-2the doubly virus liquid of dilution inoculated into chick embryo allantoic cavity respectively, 10 pieces of instar chicken embryos on the 9th～11 of every group of inoculation, every embryonic breeding kind 0.2ml, puts 33～35 DEG C and cultivates 72 hours.Dead not counting in 24 hours, every group of chicken embryo must at least survive 80%, in the chicken embryo of survival, every embryo is got 0.5ml allantoic fluid, after mixing by group, then a blind passage generation, 10 pieces of embryos of every group of each inoculation, every embryonic breeding kind 0.2ml,, after 72 hours, getting allantoic fluid and carry out hemagglutination test through 33～35 DEG C of cultivations, should not there is not hemagglutination in result.(chick embryo method)
Concrete outcome sees the following form:
The first-generation:
Remarks: sample 1: deactivation 24h, sample 2: deactivation 48h, sample 3: deactivation 72h, sample 4: deactivation 96h.
The s-generation
Remarks: sample 1: deactivation 24h, sample 2: deactivation 48h, sample 3: deactivation 72h, sample 4: deactivation 96h.
The third generation
Remarks: sample 1: deactivation 24h, sample 2: deactivation 48h, sample 3: deactivation 72h, sample 4: deactivation 96h.
Consistent with " three of pharmacopeia " chick embryo method result as deactivation the result with Vero cell, but sensitivity will be higher than " three of pharmacopeia " chick embryo method.This method contrasts former method and has that method is convenient, highly sensitive, high specificity, low cost and other advantages, and therefore cell method is more suitable for the deactivation checking as Vero stream of cells influenza vaccine.
Claims (1)
1. a detection method for inactivated influenza virus checking, is characterized in that: application Vero cell method detects the deactivation checking of influenza virus, has following steps:
(1) H3N2 type influenza virus (lot number is 20090731-5) is diluted to every hole and goes out 1-5 spot, be diluted to 10-7,0.5 × 10-7, with this extent of dilution inoculated into chick embryo, square vase, 6 orifice plates respectively:
Chicken embryo: 10
-7, 0.5 × 10
-7each 6 pieces of embryos, 0.2ml/ embryo;
Square vase: 10
-7, 0.5 × 10
-7each 6 holes, 0.5ml/ bottle;
6 orifice plates: 10
-7, 0.5 × 10
-7each 6 holes, 0.5ml/ hole;
Chicken embryo passed for 2 generations one to one, and detected respectively blood clotting titre, result of determination; Square vase passed for 2 generations one to one, and detected respectively blood clotting titre, result of determination; 6 porocyte culture plates pass 1 generation, plaque test, result of determination;
(2) utilize Veo cell method to detect inactivated influenza virus verification experimental verification:
T25 Tissue Culture Flask Cultivation of Vero, for subsequent use after growing up to individual layer;
By extremely former times, 10 of diluted sample to be checked
-1, 10
-2, 6 bottles of cells of each extent of dilution inoculation;
Every bottle of cell inoculation sample 1ml, outwells after 33 DEG C of absorption 60min, adds virus-culturing fluid 5ml;
33 DEG C of cultivations are gathered in the crops respectively nutrient solution after 72 hours and are done blood clotting;
Same extent of dilution harvest liquid was mixed by 1: 1, and in 2 generations of blind passage more as stated above, results sample does blood clotting;
Result: third generation blood clotting result is negative be judged to qualified, otherwise defective.
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|---|---|---|---|
| CN201310016065.0A CN103924005A (en) | 2013-01-16 | 2013-01-16 | Detection method for influenza virus inactivation test verification |
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|---|---|---|---|
| CN201310016065.0A CN103924005A (en) | 2013-01-16 | 2013-01-16 | Detection method for influenza virus inactivation test verification |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846120A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Recombinant herpes simplex virus titer determination method |
| CN105603126A (en) * | 2016-03-18 | 2016-05-25 | 福州大北农生物技术有限公司 | Inactivation test method for inactivated avian influenza vaccine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2429152A (en) * | 2005-08-19 | 2007-02-21 | Forum Bioscience Holdings Ltd | Electrochemically activated water for use as a virucide |
| CN102586195A (en) * | 2011-12-01 | 2012-07-18 | 哈药集团生物疫苗有限公司 | Method for preparing avian influenza virus and inactivated vaccine thereof with Vero passage cells |
-
2013
- 2013-01-16 CN CN201310016065.0A patent/CN103924005A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2429152A (en) * | 2005-08-19 | 2007-02-21 | Forum Bioscience Holdings Ltd | Electrochemically activated water for use as a virucide |
| WO2007020478A1 (en) * | 2005-08-19 | 2007-02-22 | Forum Bioscience Holdings Limited | Virucidal solutions |
| CN102586195A (en) * | 2011-12-01 | 2012-07-18 | 哈药集团生物疫苗有限公司 | Method for preparing avian influenza virus and inactivated vaccine thereof with Vero passage cells |
Non-Patent Citations (2)
| Title |
|---|
| 王雅华，邢钊: "《兽用生物制品技术》", 30 April 2009, 中国农业大学出版社, article "PRRSV灭活检验" * |
| 黄辉萍: "消毒剂灭活病毒效果的评价方法及其研究进展", 《国外医学病毒学分册》, vol. 12, no. 2, 31 December 2005 (2005-12-31), pages 59 - 62 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846120A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Recombinant herpes simplex virus titer determination method |
| CN105603126A (en) * | 2016-03-18 | 2016-05-25 | 福州大北农生物技术有限公司 | Inactivation test method for inactivated avian influenza vaccine |
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Application publication date: 20140716 |