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The Establishment of PCR Detection Methods of Duck Egg Reduction Syndrome and Serological Epidemiological Investigation in Southwest China

Author: LuoNian
Tutor: JiangWenCan
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Duck egg syndrome PCR testing Blood clotting inhibition test Serumepidemiological investigation
CLC: S858.32
Type: Master's thesis
Year: 2013
Downloads: 22
Quote: 0
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Abstract


Egg Drop Syndrome (EDS76) is caused by avian adenovirus group II] while this viral infection can make Egg production serious decline.since1976, Dutch scientists Van Eck at the first time found reduced Egg Syndrome virus (EDSV), many countries also have multiple strains of virus was isolated in the chicken. Most scholars believe that the infectious susceptible animals are chicken and duck as only the storage reservoir of the virus, but since in1984at the first time this virus is isolated by Bartha from the performance for duck egg drop disease and severe diarrhea, form that on, more and more research of duck egg reduction syndrome was represent at home and abroad, some scholars suggests that under certain conditions duck EDS76virus infection can also cause serious drop of egg production. In this study, Using duck blood serum of different parts of the three provinces from the southwest Yun, Gui, Chuan do EDSV blood clotting inhibition antibody test and leukocyte DNA-PCR test, EDSV antigens was to investigate infection status of duck egg reduction syndrome in southwest China, therefore, this study can reduce egg syndrome virus prevention monitoring in southwest and provide important basis for molecular biology research and the development of DNA vaccines.1. The establishment of duck egg reduction syndrome PCR detection methodIn this study, we design an280bp primers refer to GenBank source of duck egg reduction syndrome virus isolates127(Y09598)100k protein gene of conservative district through to the magnesium2+concentration, primer concentration, concentration of Taq polymerase, template, denaturation temperature, annealing temperature, the amount of stretching temperature, cycle times are optimized for PCR optimum reaction model.25μL reaction system, respectively, included10x2.5μL PCR Buffer L,25tendency for2μL/L MgC12, dNTPs tendency (2.5L)2μL I primers, primer Ⅱ (concentration for10pmol/μL)1μL, Tap enzyme0.5μL,2μL template DNA, plus ddH2O to25μL. Response procedures for94℃for5min,94℃to55℃,30s,30s72℃for1min,31 out of the running cycle, the extension at72℃for7min. With Duck viral Hepatitis Virus (DHV), Duck source pathogenic e. coli bacterium, Duck source pathogenic Salmonella, Duck plague creamer’s coli (Riemerella anatipestifer, RA) for the specificity of the contrast experiment and the mother Duck against poison detecting its organization with poison is proved that the detection method is effective, and has high specificity. Sensitivity experiment results show that the method can detect the minimum concentration of DNA template is14.25pg/mL, its sensitivity can be used for the clinical rapid detection of duck eggs syndrome virus and to apply this method to the EDSV detection in southwest. Through the PCR test, duck blood sample results show duck positive rate in meat type duck and eggs were18.1%and21.6%respectively, the positive rate is higher, and the duck are the storage reservoir of EDSV basic consistent, therefore, the theory of duck egg reduction in southwest syndrome defence provides data reference and theoretical guidance. We will detect the positive samples by a randomly selected form each province and do bioinformatics analysis after sequencing the genes and amino acids, the results show that the homology of100k protein biological characteristics is as high as98%above comparing with egg duck reduction syndrome virus127isolates (Y09598).2. The serum epidemiological investigation of duck egg reduction syndrome in southwest ChinaThe capital of each province were collected in major acquisition in southwest Yunnan, Guizhou, Sichuan three provinces while on behalf of southeast northwest four areas of five points. In each collection point collect10or more form Type meat and Type egg duck. Collecting16areas of a total193cases meat-type duck blood and199cases eggs-type duck blood samples and using blood clotting inhibition test on the392samples for testing. Results show that the meat ducks and egg duck positive number were22and29respectively, and the positive rate was11.4%and14.6%respectively, egg duck was susceptible to the virus comparing with meat duck. From those three provinces, concluded that type egg duck of sichuan province has the highest positive rate, type meat duck in yunnan region has the highest positive rate, this illustrate the three provinces should strengthen epidemic prevention of the disease and also to various areas duck egg reduction syndrome epidemic prevention against to some extent. By tapping the poison after different time of mother duck antibody and antigen detection, the mother duck antibody levels after infection EDSV and poisoned group have certain understanding, the results show that the poison attack after7days the HI antibody titer higher, peaked to14days and down to a certain extent in21days, HA test shows that no antigen was detected in other organization in addition to heart while EDSV infection in7-21days after the duck for detoxification achieve peak. So this study can provide a scientific basis for duck eggs reduction clinical syndrome vaccine research and development of clinical application.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Duck
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