CN104726415A - Method for isolating and culturing serum duck hepatitis A virus-3 through chick embryo - Google Patents
Method for isolating and culturing serum duck hepatitis A virus-3 through chick embryo Download PDFInfo
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Abstract
The invention relates to a method for isolating and culturing serum duck hepatitis A virus-3 (DHAV-3) through a chick embryo. DHAV-3 can cause hepatitis in ducklings. Although the virus can be isolated and cultured through a duck embryo, the virus is difficult to culture and isolate through a conventional chick embryo allantoic cavity vaccination method. According to the invention, through a method of inoculating a specific pathogen free (SPF) chick embryo yolk sac, the DHAV-3 is isolated from a diseased duck material, and cultured and passaged through the SPF chick embryo yolk sac. The method provided by the invention lays a foundation for isolating and culturing DHAV-3 as well as developing attenuated live vaccines.
Description
Technical field
The present invention relates to a kind of method of chicken embryo separation and Culture Serotype-3 DHAV (DHAV-3), belong to microbial technology field.
Background technology
DHAV (duck hepatitis A virus, DHAV) belong to microRNA Viraceae member (Lu Chengping. veterinary microbiology. (the 5th edition) Beijing: Chinese agriculture press, 2012,460-466.), it is the most important cause of disease causing duckling hepatitis, duckling below 4 week age of main infection occurs with the hemorrhage acute hepatitis for feature of liver, and morbidity is anxious, it is fast to propagate, M & M can reach more than 90%.The duck hepatitis caused by DHAV from last century the fifties after the U.S. is found, support rapidly the outburst of duck area in the world and spread, China's Huang is all built (Huang Junjian. viral hepatitis of duckling is studied, animal and veterinary institute of Shanghai Academy of Agricultural Sciences reprint, 1963.) reported first in 1963 duck field, District of Shanghai breaks out that this is sick, king's equality (king's equality. the research of Beijing viral hepatitis of duckling. Peking University's journal (natural science edition), 1980, 1:55) be first separated to virus in morbidity duck field, Beijing area in 1980, duck area is supported by China afterwards all the generation of this disease and popular.Along with succeeding in developing and clinical application of duck hepatitis attenuated live vaccines in recent years and high immunity yolk antibody, this disease is controlled to a great extent preferably.
(the Sandhu such as Sandhu in 1992, T.S., B.W.Calnek, and L.Zeman.1992.Pathologic and serologiccharacterization of a variant of duck hepatitis type I virus.Avian Dis 36:932-936.) report that duck hepatitis virus morphs phenomenon, the good grade of Su Jing in 2002 (Su Jingliang etc. the separation of New Type Duck Hepatitis Virus and preliminary evaluation. Chinese animal doctor's science and technology, 2002, 1:15-16.) report that duck hepatitis vaccine is crossed in the immunity of the area such as Beijing and Guangxi or duck hepatitis still occurs for the duck group that injected duck hepatitis high immunity yolk antibody, and prove the duck hepatitis virus serum-free cross reaction of duck hepatitis virus and the classics be separated to, therefore claim New Type Duck Hepatitis Virus temporarily.2007, Taiwan's scholars Tseng (Tseng C.H., Tsai H.J., Molecularcharacterization of a new serotype of duck hepatitis virus.Virus Res, 2007, 126:19-31.) with Korea S scholar Kim (Kim M.C., Kwon Y.K., Joh S.J., et al.Recent Korean isolates of duck hepatitis virus revealedthe presence of a new geno-and serotype when compared to duck hepatitis virus type1 typestrains.Arch Virol.2007, 152:2059-2072.) report the New Type Duck Hepatitis Virus being separated to and being different from classical duck hepatitis virus respectively, by molecular biology and serological identification, show that the New Type Duck Hepatitis Virus that Taiwan is separated with Korea S all belongs to DHAV, but also there is obvious serology difference between them, so clearly DHAV is divided into now 3 serotypes (Lu Chengping. veterinary microbiology. (the 5th edition) Beijing: Chinese agriculture press, 2012, 460-466.), and by the duck hepatitis virus of classics, Taiwan New Type Duck Hepatitis Virus and Korean Utility duck hepatitis virus belong to serum 1 type DHAV (DHAV-1) respectively, among serum 2 type DHAV (DHAV-2) and Serotype-3 DHAV (DHAV-3).Confirmed by molecular biology identification and serological test, China mainland is the New Type Duck Hepatitis Virus strain of institute's isolation identification so far and Korean Utility duck hepatitis virus very high homology from 2002, genus Serotype-3 DHAV (DHAV-3).
As everyone knows, Shi Yang duck big country of China, the year number of animals raised of current duck has reached 4,000,000,000, account for more than 70% of whole world duck raising total amount, duck hepatitis is the No.1 epidemic disease that duck production is supported by China always, applying in recent years along with serum 1 type duck hepatitis A living vaccine and high immunity yolk antibody, serum 1 type duck hepatitis A is effectively controlled, but nearly ten years owing to newly occurring that Serotype-3 duck hepatitis A supports the outburst of duck area and rapid spread in China, become again restriction China and support duck and produce new Tough questions.
Classical duck hepatitis virus, the serum 1 type DHAV (DHAV-1) namely claimed now, not only suitable duck embryo separation and Culture virus, and be separated by traditional chick embryo allantoic cavity vaccination ways and cultivate virus.And the New Type Duck Hepatitis Virus of China's discovery in recent years, i.e. Serotype-3 DHAV (DHAV-3), although can be separated by duck embryo and cultivate virus, be difficult to cultivate and be separated this virus by the method for conventional chick embryo allantoic cavity inoculation.If DHAV-3 can not adapt to chick embryo culture, just imply that development DHAV-3 living vaccine exists huge technical bottleneck.
Summary of the invention
The object of the invention is a kind of method providing chicken embryo separation and Culture Serotype-3 duck hepatitis virus (DHV-3), for the development of DHV-3 separation and Culture, attenuated live vaccines lays the foundation.
The present invention can be achieved through the following technical solutions:
A method of chicken embryo separation and Culture DHAV-3, is characterized in that the method comprises the following steps:
1. choose and on duck embryo, carry out neutralization test through anti-DHAV-1 positive serum and anti-DHAV-3 positive serum and cross neutralization test is diagnosed as the liver organization pathological material of disease of DHAV-3 for chick embryo culture isolated viral;
2.DHAV-3 organizes the process of pathological material of disease: the liver organization getting a little DHAV-3, does 1:5 (W/V) left and right dilution, grinding, freeze thawing 2 ~ 3 times, centrifugal, get centrifuged supernatant through 0.22 μm of filter Sterile Filtration with sterile saline;
3. egg inoculation isolated viral: get above-mentioned bacteria-free filtrate by yolk sac inoculation approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, 37 DEG C of cultivations, in 24h, dead germ abandons it, Continuous Observation is to 120h, if have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight;
4. chicken embryo goes down to posterity inoculation: take out idiosome in time, grind to form homogenate, centrifuging and taking supernatant liquor through 0.22 μm of Sterile Filtration, get filtrate through yolk sac inoculation 6 ~ 8 age in days SPF chicken embryo 5 ~ 10 pieces, every embryo 0.2 ~ 0.5ml; In blind passage 5 ~ 10 generation, along with the increase of passage number, the chicken embryo death time moves forward, and Embryo mortality also reaches 100% gradually, shows by the success of chicken embryo isolated viral, and adapts to chick embryo culture gradually.
The detailed description of invention:
1. get clinical typical duck hepatitis symptom to die of illness duck, aseptic technique is got the tissues such as liver and is identified that DHAV-3 is positive sample through PCR, and virus can be separated to by lethal duck embryo through 10 ~ 12 age in days duck embryo allantoic cavity inoculations, on duck embryo, carry out neutralization test and cross neutralization test with anti-DHAV-1 positive serum and anti-DHAV-3 positive serum again, be diagnosed as the liver organization pathological material of disease of DHAV-3 for chick embryo culture isolated viral.
2. organize the process of pathological material of disease: the duck liver organization pathological material of disease getting a little DHAV-3, be 1:5 (W/V) left and right dilution, grinding, freeze thawing 2 ~ 3 times, the centrifugal 10min of 3000r/min with sterile saline, get centrifuged supernatant and go bacterium to filter through 0.22um filter.
3. egg inoculation isolated viral: get above-mentioned bacteria-free filtrate by yolk sac inoculation approach inoculation 6 ~ 8 age in days SPF chicken embryos, inoculation working method is shown in Fig. 1, every embryonic breeding kind 0.2 ~ 0.5ml, each inoculation 5 ~ 10 pieces of chicken embryos, 37 DEG C of incubators are cultivated, chicken embryo death situation is observed in timing, and in 24h, dead germ abandons it, and Continuous Observation is to 120h.If have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight.
4. chicken embryo goes down to posterity inoculation: take out above-mentioned refrigeration chicken embryo aseptic technique in time and take out idiosome, and observe idiosome pathology situation, chicken embryo is ground to form homogenate, the centrifugal 10min of 3000r/min, get centrifuged supernatant and go bacterium to filter through 0.22 μm of filter.By bacteria-free filtrate again by yolk sac inoculation approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, each inoculation 5 ~ 10 pieces of chick embryo culture, there is not death or sporadic deaths in the initial chicken embryo in 120h that goes down to posterity several times, but after chick embryo yolk sac inoculation blind passage 5 ~ 10 generation, then visible chicken embryo starts in 120h to occur dead after inoculation, along with the increase of passage number, the chicken embryo death time moves forward, gradually in concentrated 48 ~ 96h after inoculation, Embryo mortality also reaches 100% gradually, show by the success of chicken embryo isolated viral, and adapt to chick embryo culture gradually.
The Microbial resources information that the present invention relates to
The microorganism that the present invention relates to has: Serotype-3 DHAV (duck hepatitis A virus, DHAV-3) HuB strain, be separated to by Hubei that (this strain continues through chicken embryo passaged virus strain virulence attenuation of and obtains the weak viral disease strain preparing vaccine, called after Serotype-3 DHAV (duck hepatitis A virus, DHAV-3) HuB60 strain, this strain delivered China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 01 06th, 2015, preserving number CGMCC No.10307), Serotype-3 DHAV (duck hepatitis A virus, DHAV-3) SDLY strain is assigned to by Shandong, Serotype-3 DHAV (duckhepatitis A virus, DHAV-3) JSXZ strain is separated to by Jiangsu, Serotype-3 DHAV (duck hepatitisA virus, DHAV-3) HeBHD strain effluent north is separated to.
Accompanying drawing explanation
Fig. 1 chick embryo yolk sac inoculation schematic diagram
Positive effect of the present invention
The present invention relates to a kind of method of chicken embryo separation and Culture DHAV-3.The duck hepatitis that DHAV-3 causes, although can be separated by duck embryo and cultivate virus, but be difficult to cultivate and be separated this virus by the method for conventional chick embryo allantoic cavity inoculation, the present invention adopts inoculation specific pathogen free (SPF) chick embryo yolk sac to isolate DHV-3 by ill duck pathological material of disease, and undertaken cultivating and going down to posterity by SPF chick embryo yolk sac, for the development of Serotype-3 duck hepatitis virus separation and Culture, attenuated live vaccines lays the foundation.
Embodiment
Embodiment 1
---DHAV-3HuB isolation of strains
The clinical generation in duck field, Hubei typical case duck hepatitis symptom 5 age in days is died of illness duck, aseptic technique gets liver organization through laboratory qualification: (1) RT-PCR detects: get hepatic tissue sample extraction RNA, through pcr amplification and electrophoresis, there is DHAV-3 specific band; (2) virus purification: get a little liver organization, 1: 10 (W/V) dilution, grinding, freeze thawing 2 times, the centrifugal 10min of 3000r/min is with every milliliter of sterile saline containing each 1000 units of penicillin and streptomycin, getting centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, get this filtrate and inoculate the responsive duck embryo 5 of 11 ages in days by allantoic cavity approach, every embryonic breeding kind 0.2ml, 37 DEG C of incubators are cultivated, and after inoculation, 48 ~ 96h duck embryo is all dead.(3) serum neutralization test qualification: get dead duck embryo allantoic liquid 100 times dilution, after anti-DHAV-1 positive serum and anti-DHAV-3 positive serum effect 1h, duck embryo carries out serum neutralization test and cross neutralization test, in table 1 with equivalent respectively.Laboratory detection result shows, the duck hepatitis of the clinical generation in duck field, Hubei is caused by DHAV-3.
Table 1 serum neutralization test and cross neutralization test result
Chick embryo yolk sac inoculation isolated viral: get the liver organization making a definite diagnosis infection DHAV-3 a little, be 1: 5 (W/V) dilution, grinding, freeze thawing 3 times, the centrifugal 10min of 3000r/min with sterile saline, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter.Get this bacteria-free filtrate and inoculate 6 age in days SPF chicken embryos by yolk sac approach.Every embryonic breeding kind 0.2ml, inoculates 10 pieces of chicken embryos, and 37 DEG C of incubators are cultivated, and chicken embryo death situation is observed in timing, and in 24h, dead germ abandons it, and Continuous Observation is to 120h.If have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight.Chicken embryo goes down to posterity inoculation: take out idiosome to above-mentioned refrigeration chicken embryo aseptic technique, and observe idiosome pathology situation, chicken embryo is ground to form homogenate, the centrifugal 10min of 3000r/min, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, by bacteria-free filtrate again by yolk sac approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, each inoculation 5 ~ 10 pieces of chick embryo culture, so by chick embryo yolk sac inoculation blind passage to 9 generation, chicken embryo 100% can be caused dead in 120h, show by the success of chicken embryo isolated viral, and name the DHAV-3 of this separation to be HuB strain, HuB strain chicken embryo goes down to posterity separating resulting in table 2.
The chicken embryo of table 2 HuB strain goes down to posterity situation
(note: this strain continues through chicken embryo passaged virus strain virulence attenuation of and obtains the weak viral disease strain preparing vaccine, called after Serotype-3 DHAV (duck hepatitis A virus, DHAV-3) HuB60 strain, this strain delivered China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 01 06th, 2015, preserving number CGMCC No.10307).
Embodiment 2
---DHAV-3SDLC isolation of strains
The clinical generation in Distributions in Liaocheng of Shandong Province duck field typical case duck hepatitis symptom 7 age in days is died of illness duck, aseptic technique gets liver organization through laboratory qualification: (1) RT-PCR detects: get hepatic tissue sample extraction RNA, through pcr amplification and electrophoresis, there is DHAV-3 specific band; (2) virus purification: get a little liver organization, 1: 5 (W/V) dilution, grinding, freeze thawing 3 times, the centrifugal 10min of 3000r/min is with every milliliter of sterile saline containing each 1000 units of penicillin and streptomycin, getting centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, get this filtrate and inoculate the responsive duck embryo 5 of 11 ages in days by allantoic cavity approach, every embryonic breeding kind 0.2ml, 37 DEG C of incubators are cultivated, and after inoculation, 48 ~ 72h duck embryo is all dead.(3) serum neutralization test qualification: get dead duck embryo allantoic liquid 100 times dilution, after equivalent anti-DHAV-1 positive serum and anti-DHAV-3 positive serum effect 1h, duck embryo carries out serum neutralization test and cross neutralization test, in table 3.Laboratory detection result shows, the duck hepatitis of the clinical generation in Distributions in Liaocheng of Shandong Province duck field is caused by DHAV-3.
Table 3 serum neutralization test and cross neutralization test result
Chick embryo yolk sac inoculation isolated viral: get the liver organization making a definite diagnosis infection DHAV-3 a little, be 1: 10 (W/V) dilution, grinding, freeze thawing 3 times, the centrifugal 10min of 3000r/min with sterile saline, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter.Get this bacteria-free filtrate and inoculate 6 age in days SPF chicken embryos by yolk sac approach.Every embryonic breeding kind 0.3ml, inoculates 10 pieces of chicken embryos, and 37 DEG C of incubators are cultivated, and chicken embryo death situation is observed in timing, and in 24h, dead germ abandons it, and Continuous Observation is to 120h.If have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight.Chicken embryo goes down to posterity inoculation: take out idiosome to above-mentioned refrigeration chicken embryo aseptic technique, and observe idiosome pathology situation, chicken embryo is ground to form homogenate, the centrifugal 10min of 3000r/min, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, by bacteria-free filtrate again by yolk sac approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, each inoculation 5 ~ 10 pieces of chick embryo culture, so by chick embryo yolk sac inoculation blind passage to 12 generation, chicken embryo 100% can be caused dead in 120h, show by the success of chicken embryo isolated viral, and name the DHAV-3 of this separation to be SDLC strain, SDLC strain chicken embryo goes down to posterity separating resulting in table 4.
The chicken embryo of table 4 SDLC strain goes down to posterity situation
Embodiment 3
---DHAV-3JSXZ isolation of strains
The clinical generation in duck field, Xuzhou typical case duck hepatitis symptom 8 age in days is died of illness duck, aseptic technique gets liver organization through laboratory qualification: (1) RT-PCR detects: get hepatic tissue sample extraction RNA, through pcr amplification and electrophoresis, there is DHAV-3 specific band; (2) virus purification: get a little liver organization, 1: 5 (W/V) dilution, grinding, freeze thawing 2 times, the centrifugal 10min of 3000r/min is with every milliliter of sterile saline containing each 1000 units of penicillin and streptomycin, getting centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, get this filtrate and inoculate 10 ages in days responsive duck embryo embryo 5 by allantoic cavity approach, every embryonic breeding kind 0.2ml, 37 DEG C of incubators are cultivated, and after inoculation, 24 ~ 72h duck embryo is all dead.(3) serum neutralization test qualification: get dead duck embryo allantoic liquid 100 times dilution, after equivalent anti-DHAV-1 positive serum and anti-DHAV-3 positive serum effect 1h, duck embryo carries out serum neutralization test and cross neutralization test, in table 5.Laboratory detection result shows, the duck hepatitis of the clinical generation in duck field, Xuzhou is caused by DHAV-3.
Table 5 serum neutralization test and cross neutralization test result
Chick embryo yolk sac inoculation isolated viral: get the liver organization making a definite diagnosis infection DHAV-3 a little, be 1: 10 (W/V) dilution, grinding, freeze thawing 3 times, the centrifugal 10min of 3000r/min with sterile saline, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter.Get this bacteria-free filtrate and inoculate 6 age in days SPF chicken embryos by yolk sac approach.Every embryonic breeding kind 0.3ml, inoculates 10 pieces of chicken embryos, and 37 DEG C of incubators are cultivated, and chicken embryo death situation is observed in timing, and in 24h, dead germ abandons it, and Continuous Observation is to 120h.If have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight.Chicken embryo goes down to posterity inoculation: take out idiosome to above-mentioned refrigeration chicken embryo aseptic technique, and observe idiosome pathology situation, chicken embryo is ground to form homogenate, the centrifugal 10min of 3000r/min, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, by bacteria-free filtrate again by yolk sac approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, each inoculation 5 ~ 10 pieces of chick embryo culture, so by chick embryo yolk sac inoculation blind passage to 15 generation, chicken embryo 100% can be caused dead in 120h, show by the success of chicken embryo isolated viral, and name the DHAV-3 of this separation to be JSXZ strain, JSXZ strain chicken embryo goes down to posterity separating resulting in table 6.
The chicken embryo of table 6 JSXZ strain goes down to posterity situation
Embodiment 4
---DHAV-3HeBHD isolation of strains
The clinical generation in duck field, Handan, Hebei typical case duck hepatitis symptom 5 age in days is died of illness duck, aseptic technique gets liver organization through laboratory qualification: (1) RT-PCR detects: get hepatic tissue sample extraction RNA, through pcr amplification and electrophoresis, there is Serotype-3 DHAV specific band; (2) virus purification: get a little liver organization, 1: 10 (W/V) dilution, grinding, freeze thawing 2 times, the centrifugal 10min of 3000r/min is with every milliliter of sterile saline containing each 1000 units of penicillin and streptomycin, getting centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, get this filtrate and inoculate the responsive duck embryo 5 of 12 ages in days by allantoic cavity approach, every embryonic breeding kind 0.2ml, 37 DEG C of incubators are cultivated, and after inoculation, 48 ~ 96h duck embryo is all dead.(3) serum neutralization test qualification: get dead duck embryo allantoic liquid 100 times dilution, after equivalent anti-DHAV-1 positive serum and anti-DHAV-3 positive serum effect 1h, duck embryo carries out serum neutralization test and cross neutralization test, in table 7.Laboratory detection result shows, the duck hepatitis of the clinical generation in duck field, Handan, Hebei is caused by DHAV-3.
Table 7 serum neutralization test and cross neutralization test result
Chick embryo yolk sac inoculation isolated viral: get the liver organization making a definite diagnosis infection DHAV-3 a little, be 1: 5 (W/V) dilution, grinding, freeze thawing 2 times, the centrifugal 10min of 3000r/m with sterile saline, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter.Get this bacteria-free filtrate and inoculate 6 age in days SPF chicken embryos by yolk sac approach.Every embryonic breeding kind 0.3ml, inoculates 10 pieces of chicken embryos, and 37 DEG C of incubators are cultivated, and chicken embryo death situation is observed in timing, and in 24h, dead germ abandons it, and Continuous Observation is to 120h.If have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight.Chicken embryo goes down to posterity inoculation: take out idiosome to above-mentioned refrigeration chicken embryo aseptic technique, and observe idiosome pathology situation, chicken embryo is ground to form homogenate, the centrifugal 10min of 3000r/min, centrifuged supernatant goes bacterium to filter through 0.22 μm of filter, by bacteria-free filtrate again by yolk sac approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, each inoculation 5 ~ 10 pieces of chick embryo culture, so by chick embryo yolk sac inoculation blind passage to 10 generation, chicken embryo 100% can be caused dead in 120h, show by the success of chicken embryo isolated viral, and name the DHAV-3 of this separation to be HeBHD strain, HeBHD strain chicken embryo goes down to posterity separating resulting in table 8.
The chicken embryo of table 8 HeBHD strain goes down to posterity situation
Claims (1)
1. a method for chicken embryo separation and Culture Serotype-3 DHAV (DHAV-3), is characterized in that the method comprises the following steps:
(1) choose and on duck embryo, carry out neutralization test through antiserum(antisera) 1 type DHAV (DHAV-1) positive serum and anti-DHAV-3 positive serum and cross neutralization test is diagnosed as the liver organization pathological material of disease of DHAV-3 for chick embryo culture isolated viral;
(2) DHAV-3 organize the process of pathological material of disease: the liver organization getting a little DHAV-3, do 1:5 (W/V) left and right dilution, grinding, freeze thawing 2 ~ 3 times, centrifugal with sterile saline, get centrifuged supernatant through 0.22 μm of filter Sterile Filtration;
(3) egg inoculation isolated viral: get above-mentioned bacteria-free filtrate by yolk sac inoculation approach inoculation 6 ~ 8 age in days SPF chicken embryos, every embryonic breeding kind 0.2 ~ 0.5ml, 37 DEG C of cultivations, in 24h, dead germ abandons it, Continuous Observation is to 120h, if have chicken embryo death therebetween, take out at any time and put 2 ~ 8 DEG C of refrigerator cold-storage a few hours or spend the night; If without death, then get 3 pieces of embryos at random in 72 ~ 96h and put 2 ~ 8 DEG C of refrigerated overnight;
(4) chicken embryo goes down to posterity inoculation: take out idiosome in time, grind to form homogenate, centrifuging and taking supernatant liquor through 0.22 μm of Sterile Filtration, get filtrate through yolk sac inoculation 6 ~ 8 age in days SPF chicken embryo 5 ~ 10 pieces, every embryo 0.2 ~ 0.5ml; In blind passage 5 ~ 10 generation, along with the increase of passage number, the chicken embryo death time moves forward, and Embryo mortality also reaches 100% gradually, shows by the success of chicken embryo isolated viral, and adapts to chick embryo culture gradually.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102174476A (en) * | 2010-12-29 | 2011-09-07 | 中国科学院微生物研究所 | Inactivated vaccine for preventing duck virus hepatitis and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174476A (en) * | 2010-12-29 | 2011-09-07 | 中国科学院微生物研究所 | Inactivated vaccine for preventing duck virus hepatitis and preparation method thereof |
| CN103382507A (en) * | 2013-06-08 | 2013-11-06 | 四川农业大学 | One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof |
Non-Patent Citations (2)
| Title |
|---|
| MIN-CHUL KIM 等: ""Development of duck hepatitis A virus type 3 vaccine and its use to protect ducklings against infections"", 《VACCINE》 * |
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Application publication date: 20150624 |