CN102749427B - A kind of method for testing efficacy of duck hemorrhagic oaritis inactivated vaccine - Google Patents
A kind of method for testing efficacy of duck hemorrhagic oaritis inactivated vaccine Download PDFInfo
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Abstract
The invention provides a kind of method for testing efficacy of duck hemorrhagic oaritis inactivated vaccine, the method uses duck hemorrhagic oaritis inactivated vaccine immune duck, immunity 2 times, carries out two for 2 weeks after head exempts from and exempts from altogether, and two exempt to attack poison by duck hemorrhagic oaritis virus to immune duck in latter 3 ~ 5 weeks; Duck serum is gathered after attacking poison, the serum be separated is inoculated SPF chicken embryo or duck embryo, calculate dead in 24-168h and the embryo number of duck hemorrhagic oaritis virus-positive, control group duck virus purification positive rate is more than 80% and the virus purification negative rate of immune group duck is more than 70% be judged to vaccine potency and be up to the standards.The method for testing efficacy of duck hemorrhagic oaritis inactivated vaccine provided by the invention has easy and simple to handle, result advantage accurately, can be applicable.
Description
Technical field
The present invention relates to a kind of method for testing efficacy of inactivated vaccine for animals, particularly relate to a kind of method of inspection of duck hemorrhagic oaritis inactivated vaccine effect, belong to veterinary biologics inspection technology field.
Background technology
Duck hemorrhagic oaritis (DuckHemorrhagicOvaritis, DH) be a kind of Novel duck infectious disease of popular, acute, high degree in contact recently, its cause of disease is duck hemorrhagic oaritis virus (DuckHemorrhagicOvaritisVirus, DHOV), virus taxis belongs to the member of flaviviridae, infect different days duck, cause the ovarian hemorrhage of sexal maturity duck, orchiatrophy, egg production, fertility rate and hatchability to decline, duck death rate increases.
In the research of virus taxis status, by carrying out sequence homology analysis and evolutionary analysis to the F4 of isolated viral strain for the genome sequence of duck embryo passaged virus and known virus, find that the genetic distance of the tembusu virus of this virus and flaviviridae Flavivirus is nearest.In diagnosis, Cao Zhenzhen, Wan Chun and, Su Jingliang, Teng Qiao be vast etc., and people's report can adopt the methods such as clinical symptoms, cardinal principle and histopathologic change, pcr amplification and virus purification to make a definite diagnosis this disease.About the prevention and control of this disease, the strain that Beijing City Agriculture and Forestry Institute and Ruipu (Baoding) Biological Pharmaceutical Co., Ltd., Qilu Animal Health Products Co., Ltd., Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute and animal and veterinary research institute of academy of agricultural sciences of Chongqing City are separated during reporting and utilizing this disease to break out prepares the method for vaccine.Lin Jian, Liu Yuehuan etc. report the artificial challenge's model utilizing laying ducks successfully to establish duck hemorrhagic oaritis, and experiment basis has been established in the evaluation for vaccine.There is not yet the play-by-play of the vaccine potency evaluation method of objective, feasible, strong operability and science.
Summary of the invention
One of the object of the invention is to provide age in days scope and the sex of a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation duck.
Two of the object of the invention is to provide duck hemorrhagic oaritis inactivated vaccine effect evaluation vaccine immunity number of times, employing time, the tissue samples of collection, Infection route and inspection embryo.
Three of the object of the invention is to provide a kind of duck hemorrhagic oaritis inactivated vaccine effect evaluation method.
The present invention's virus stain called after used duck hemorrhagic oaritis virus HB strain, preserving number is CCTCCV201122; Described duck hemorrhagic oaritis virus is ssRNA virus, has cyst membrane, can aggegation dove red blood cell; Virion is spherical substantially, diameter 40 ~ 60nm; The resistibility of described virus environment is to external world comparatively strong, and in 4 DEG C, deposit a few week, in-20 DEG C, deposit some months, its appeal is all unaffected; Described virus is responsive to ether, chloroform; Most of detergent can by its rapid inactivation; Under 37 DEG C of conditions, fumigating 6h with 0.1% formalin just can its deactivation.This virus can at 9 ~ 13 age in days duck embryo allantoic cavities, embryo CAM, 9 ~ 10 age in days chick embryo allantoic cavities, embryo CAM, and 6 age in days chick embryo yolk sacs are cultivated in 7 ~ 8 age in days duck embryo yolk bags, also can be cultivated on DEF and BHK21 cell.
The preparation method of the present invention's duck hemorrhagic used oaritis inactivated vaccine declared patent of invention in 2011, and application number is 201110255115.1.
The invention provides a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy, the method uses duck hemorrhagic oaritis inactivated vaccine immune duck, immunity 2 times, carries out two for 2 weeks after head exempts from and exempts from altogether, and two exempt to attack poison by duck hemorrhagic oaritis virus to immune duck in latter 3 ~ 5 weeks; Duck serum is gathered after attacking poison, the serum be separated is inoculated SPF chicken embryo or duck embryo, calculate dead in 24-168h and the embryo number of duck hemorrhagic oaritis virus-positive, control group duck virus purification positive rate is more than 80% and the virus purification negative rate of immune group duck is more than 70% be judged to vaccine potency and be up to the standards.
Further, the inventive method, according to neurological susceptibility experimental result, determines that duck hemorrhagic oaritis inactivated vaccine efficacy test is 120 ~ 560 ages in days with the age in days of duck.
Preferably, efficacy test is female by the sex of duck.
Wherein, the dosage of 2 immune duck hemorrhagic oaritis inactivated vaccines is 0.5ml/ ~ 2ml/.
Wherein, attacking poison duck hemorrhagic oaritis virus is DHOV-HB strain (deposit number is CCTCCV201122, has been open in the Chinese patent of 201110255115.1 at application number), and attacking toxic agent amount is 3 × 10
4eLD
50.
Wherein, inoculated into chick embryo or duck embryo are through yolk sac inoculation 6 ~ 8 age in days SPF chicken embryo or duck embryo.
Wherein, the inoculum concentration of chicken embryo or duck embryo is 0.2ml/ embryo.
Wherein, attack poison rear 4-5 day, gather duck serum.
Wherein, described duck is that meat duck, egg are with duck or SPF duck.
Further, the invention provides a kind of duck hemorrhagic oaritis inactivated vaccine method for testing efficacy, the method uses duck hemorrhagic oaritis inactivated vaccine immunity 120 ~ 560 age in days duck, and within 2 weeks, carry out two after head exempts from and exempt from, the immunizing dose of twice is only 0.5 ~ 2ml/; Two exempt from after within 3 ~ 5 weeks, attack poison with DHOV-HB strain, Infection route is oral, intramuscular injection or intravenous injection, and attacking toxic agent amount is 3 × 10
4eLD
50attack poison rear 4-5 day, gather duck serum, through yolk bag approach inoculation 6 ~ 8 age in days SPF chicken embryo or duck embryos, 0.2ml/ embryo, 37 DEG C of hatchings, calculate that embryo in 24 ~ 168h is dead and the embryo number being detected as the duck hemorrhagic oaritis virus infections positive is judged to the virus purification positive, and control group duck virus purification positive rate is more than 80% and the virus purification negative rate of immune group duck is more than 70% be judged to vaccine potency and be up to the standards.
Blood sampling time, Infection route and collecting sample: after duck hemorrhagic oaritis inactivated vaccine first immunisation, within 2 weeks, carry out secondary immunity, after secondary immunity, 3 ~ 5 weeks oral administration, muscle or intravenous routes attack poison, attack poison rear 4-5 day, blood sampling, separation of serum, is more than 80% for virus purification control group duck virus purification positive rate and the virus purification negative rate of immune group duck is more than 70% be judged to vaccine potency and be up to the standards.
The present invention proposes the age in days and the sex that use duck for duck hemorrhagic oaritis inactivated vaccine efficacy test first; the immune time of vaccine potency inspection time vaccines, Infection route, time of drawing materials, the tissue samples of collection, inspection embryo, propose the standard that the vaccine potency test method of immunoprotection-virus purification, criterion and vaccine quality are qualified.Solve the blank of duck hemorrhagic oaritis inactivated vaccine effect evaluation method and criterion.The method that the present invention determines is objective, comprehensive, strong operability, can be used for laboratory and the clinical efficacy evaluation of duck hemorrhagic oaritis inactivated vaccine.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, biology used in embodiment, chemical reagent are conventional commercial, the conventional means that technological means used in embodiment is well known to those skilled in the art.Beijing duck used in embodiment is purchased from duck hemorrhagic oaritis negative antibody duck field (Beijing Golden Star Duck Center Nan Kou kind duck field), sheldrake is purchased from duck hemorrhagic oaritis negative antibody duck field (under the Pearl Spring of four seas town, Yanqing County of Beijing county dress circle village), SPF duck is hatched by the SPF duck's egg purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center, raises to the required age in days of test in SPF chicken isolator.
The embodiment 1 duck hemorrhagic oaritis inactivated vaccine efficacy test age in days of duck
The Beijing duck of 42,50,100,120,200,300,400,560 and 600 ages in days is divided into 9 groups, often organizes 10, often group is the Beijing duck (purchased from Beijing Golden Star Duck Center Nan Kou kind duck field, duck hemorrhagic oaritis antibody is negative) of same age in days.Test duck oral route attacks the DHOV-HB strain virus of malicious same dose, about containing 3 × 10
4eLD
50/ only, attack malicious latter 5 days through wing venous collection duck blood, aseptic separation of serum.Serum inoculates 6 age in days SPF chicken embryos through yolk bag approach, 5 pieces/duck, 0.2ml/ embryo, puts 37 DEG C and continues hatching to 168h.Within 24h, dead chicken embryo discards, and calculates dead in 24 ~ 168h and the RT-PCR of DHOV detects positive chicken embryo number.1 piece and above chicken embryo death is had and the RT-PCR of DHOV detects positive that to be designated as this duck virus purification positive in every part of serum 5 pieces of chicken embryos.According to neurological susceptibility experimental result, determine that duck hemorrhagic oaritis inactivated vaccine efficacy test is 120 ~ 560 ages in days with the age in days of duck.
The test stone of susceptible duck or duck embryo
(1) susceptible duck 5 is only from without the immunity of duck hemorrhagic oaritis inactivated vaccine or have the duck of duck hemorrhagic oaritis popular history duck group, the inspection strain of every oral 1.0 milliliters of aseptic 0.5% lactoalbumin hydrolysate Hank ' s liquid 100 times dilutions of duck.Attack poison latter 5 days, blood sampling, separation of serum carries out virus purification.Virus isolated rate should at least 4/5 positive.
(2) susceptible embryo 10 pieces is from kind egg that is immune without duck hemorrhagic oaritis inactivated vaccine or that have duck hemorrhagic oaritis pestilence history kind duck group to produce, and 37 DEG C of hatchings are to 10 ages in days.Inoculate the kind poison of any generation of 0.5% aseptic lactoalbumin hydrolysate Hank ' s liquid (LH) 500 times dilution through allantoic cavity, every embryo 0.1ml, continues hatching under putting 36 ~ 37 DEG C of conditions.Duck embryo should death in 24 ~ 96 hours at least 9/10 after inoculation.It is systemic bleeding that dead duck embryo observes pathology.
The embodiment 2 duck hemorrhagic oaritis inactivated vaccine effect evaluation sex of duck
The Beijing duck oral route of 80 120 age in days male and female half and half is attacked the DHOV-HB strain virus of malicious same dose, about containing 3 × 10
4eLD
50/ only.Attack malicious latter 1 ~ 8 day through wing venous collection duck blood, 10 pcs/day, aseptic separation of serum.Serum inoculates 6 age in days SPF chicken embryos through yolk bag approach, and the serum of every duck inoculates 5 pieces of chicken embryos, 0.2ml/ embryo, puts 37 DEG C and continues hatching to 168h.Within 24h, dead chicken embryo discards, and calculates death in 24 ~ 168h and DHOV positive chicken embryo number.Have 1 piece and above chicken embryo death in every part of serum 5 pieces of chicken embryos and DHOV positive to be designated as this duck virus purification positive.Female Beijing duck virus purification positive rate is apparently higher than male Beijing duck.The positive rate of the virus infections chicken embryo that poison of attacking against each other occurs after male and female duck serum inoculated into chick embryo afterwards every day carries out the paired t inspection of two sample, and P value is less than 0.05, and there were significant differences in neurological susceptibility for sex.
Immunoprotection-the isolation of virus (1) of embodiment 3 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012001, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 140 age in days Beijing duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 1ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 4 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and oral route attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the Beijing duck (10, female) that do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible to carry out attacking poison for control group simultaneously.Attack poison latter 5th day, gather duck serum, every duck serum inoculates 5 piece of 6 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 90%, and the incidence of disease of non-immune group duck is 80%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (2) of embodiment 4 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012002, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 120 age in days Beijing duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 0.5ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 3 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and intramuscular injection path attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish simultaneously do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible Beijing duck (10, female) carry out attacking poison for control group.Attack poison latter 5th day, gather duck serum, every duck serum inoculates 5 piece of 6 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 70%, and the incidence of disease of non-immune group duck is 90%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (3) of embodiment 5 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012003, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 560 age in days Beijing duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 2ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 5 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and oral route attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the Beijing duck (10, female) that do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible to carry out attacking poison for control group simultaneously.Attack poison latter 5th day, gather duck serum, every duck serum inoculates 5 piece of 7 age in days SPF duck embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead duck embryo discards, in 24 ~ 168 hours in duck embryo the dead and RT-PCR positive of 1 piece and above duck embryo to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 80%, and the incidence of disease of non-immune group duck is 100%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (4) of embodiment 6 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012004, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 300 age in days Beijing duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 1ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 4 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and Intravenous administration route attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the Beijing duck (10, female) that do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible to carry out attacking poison for control group simultaneously.Attack poison latter 5th day, gather duck serum, every duck serum inoculates 5 piece of 8 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 70%, and the incidence of disease of non-immune group duck is 90%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (5) of embodiment 7 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012005, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 400 age in days Beijing duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 1.5ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 3 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and oral route attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the Beijing duck (10, female) that do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible to carry out attacking poison for control group simultaneously.Attack poison latter 5 days, gather duck serum, every duck serum inoculates 5 piece of 8 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 90%, and the incidence of disease of non-immune group duck is 90%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (6) of embodiment 8 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012006, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 200 age in days Beijing duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 0.5ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 5 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and intramuscular injection path attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the Beijing duck (10, female) that do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible to carry out attacking poison for control group simultaneously.Attack poison latter 5 days, gather duck serum, every duck serum inoculates 5 piece of 7 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 80%, and the incidence of disease of non-immune group duck is 90%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (7) of embodiment 9 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012001, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 160 age in days sheldrake 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 0.5ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 5 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and oral route attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the sheldrake (10, female) that do not infect DHOV and non-immune duck hemorrhagic oaritis inactivated vaccine susceptible to carry out attacking poison for control group simultaneously.Attack poison latter 4 days, gather duck serum, every duck serum inoculates 5 piece of 6 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 70%, and the incidence of disease of non-immune group duck is 80%, illustrates that this batch of vaccine potency is up to the standards.
Immunoprotection-the isolation of virus (8) of embodiment 10 duck hemorrhagic oaritis inactivated vaccine effect evaluation
Use duck hemorrhagic oaritis inactivated vaccine (lot number 2012001, Beijing City Agriculture and Forestry Institute Raise Livestockv Veterinarian Institute and Ruipu's development) immune 120 age in days SPF duck 10, be female, as vaccine immunity group.The immunizing dose of vaccine immunity group and method are: it is 0.5ml/ that head exempts from vaccine dose, and chest muscle is injected.Head exempts from 2 weeks afterwards, exempts from identical approach two according to same dose, and two exempt within 5 weeks, to attack poison afterwards, and attacking poison strain is DHOV-HB strain, and oral route attacks poison, and every duck is attacked toxic agent amount and is about 3 × 10
4eLD
50.Establish the SPF duck (10, female) that do not infect DHOV to carry out attacking poison for control group simultaneously.Attack poison latter 4 days, gather duck serum, every duck serum inoculates 5 piece of 6 age in days SPF chicken embryo through yolk bag approach, 0.2ml/ embryo, 37 DEG C of hatchings, Continuous Observation to 168 hour.In 24 hours, dead chicken embryo discards, in 24 ~ 168 hours in chicken embryo 1 piece and above chicken embryo death the RT-PCR positive to be calculated as this duck hemorrhagic oaritis virus purification positive.The malicious protection ratio of attacking of immune group duck is 80%, and the incidence of disease of non-immune group duck is 90%, illustrates that this batch of vaccine potency is up to the standards.
Embodiment of the present invention 3-10 all uses the method for RT-PCR to detect virus, the NS5 gene design Flavivirus universal primer (upstream: 5 '-TCAAGGAACTCCACACATGAGATGTACT-3 ' of the primer foundation bibliographical information of the method; Downstream: 5 '-GTGTCCCATCCTGCTGTGTCATCAGCATACA-3 '), theoretical amplification fragment length 988bp.
The RNA carrying out chick embryo allantoic liquid or idiosome according to Trizol kit instructions extracts, establish positive and negative quality-control sample simultaneously, RT-PCR is carried out according to kit instructions, PCR primer gets the agarose gel electrophoresis that 10 μ l carry out 1%, amplified fragments is at about 1Kb, and positive control is at about 1Kb, negative control about 1Kb occurs without band, then can judge that testing sample is duck hemorrhagic oaritis virus-positive.
Claims (1)
1. a duck hemorrhagic oaritis inactivated vaccine method for testing efficacy, is characterized in that, use duck hemorrhagic oaritis inactivated vaccine immunity 120 ~ 560 age in days duck, within 2 weeks, carry out two after head exempts from and exempt from, the immunizing dose of twice is only 0.5 ~ 2ml/; Two exempt from after within 3 ~ 5 weeks, attack poison with DHOV-HB strain, Infection route is oral, intramuscular injection or intravenous injection, and attacking toxic agent amount is 3 × 10
4eLD
50after attacking malicious 4-5 day, gather duck serum, through yolk bag approach inoculation 6 ~ 8 age in days SPF chicken embryo or duck embryos, 0.2ml/ embryo, 37 DEG C of hatchings, calculate that embryo in 24 ~ 168h is dead and the embryo number being detected as the duck hemorrhagic oaritis virus infections positive is judged to the virus purification positive, judge that whether the inspection of vaccine potency is qualified by the virus purification negative rate of control group duck virus purification positive rate and immune group duck.
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| CN102302772A (en) * | 2011-08-31 | 2012-01-04 | 齐鲁动物保健品有限公司 | Duck hemorrhagic ovaritis (DHO) inactivated vaccine and preparation method thereof |
| CN102329782A (en) * | 2011-08-16 | 2012-01-25 | 福建省农业科学院畜牧兽医研究所 | Avian flavivirus isolation strain and application thereof in preparation of vaccine |
| CN102488893A (en) * | 2011-12-28 | 2012-06-13 | 瑞普（保定）生物药业有限公司 | Duck hemorrhagic oophoritis inactivated vaccine production method by using cell line and product thereof |
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| CN102220291A (en) * | 2011-05-18 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Duck flavivirus and inactivated vaccine thereof |
| CN102304494B (en) * | 2011-08-31 | 2013-04-17 | 北京市农林科学院畜牧兽医研究所 | Duck hemorrhagic ovaritis virus strain, inactivated vaccine and preparation method thereof |
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| CN102329782A (en) * | 2011-08-16 | 2012-01-25 | 福建省农业科学院畜牧兽医研究所 | Avian flavivirus isolation strain and application thereof in preparation of vaccine |
| CN102302772A (en) * | 2011-08-31 | 2012-01-04 | 齐鲁动物保健品有限公司 | Duck hemorrhagic ovaritis (DHO) inactivated vaccine and preparation method thereof |
| CN102488893A (en) * | 2011-12-28 | 2012-06-13 | 瑞普（保定）生物药业有限公司 | Duck hemorrhagic oophoritis inactivated vaccine production method by using cell line and product thereof |
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