本文關(guān)鍵詞： 雞傳染性貧血病毒 禽腺病毒 弱毒疫苗 SYBR GreenⅠ熒光定量PCR PCR 污染　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：雞傳染性貧血病(Chicken infectious anemia,CIV)是由雞傳染性貧血病毒(Chicken infectious anemia virus,CIAV)引起的雛雞發(fā)生再生障礙性貧血,以全身性淋巴組織萎縮為特點(diǎn)的免疫抑制性傳染病。禽腺病毒(Fowl adenovirus,FAdV)可引發(fā)雞產(chǎn)生包涵體肝炎(IBH)、心包積液綜合征(HPS)和肌胃糜爛(GE)等疾病。CIAV和FAdV均可造成感染雞群處于免疫抑制狀態(tài),使雞群對其他病原易感性增強(qiáng),同時(shí)對馬立克、傳染性法氏囊病及禽網(wǎng)狀內(nèi)皮增生病等疾病的疫苗保護(hù)力降低,從而導(dǎo)致雞群在共感染或繼發(fā)感染等其他病原時(shí)死亡率升高,對肉雞產(chǎn)業(yè)和SPF雞蛋的生產(chǎn)造成嚴(yán)重的經(jīng)濟(jì)損失。CIAV和FAdV除垂直傳播和水平傳播外,同時(shí)還可以通過疫苗污染途徑傳播,因此本研究建立了用于疫苗中監(jiān)測CIAV和FAdV的快速檢測方法,將有助于企業(yè)能夠準(zhǔn)確而快速地檢測禽弱毒疫苗中的CIAV和FAdV污染。一、CIAV SYBR Green檢測方法的建立及其在疫苗污染檢測中的應(yīng)用根據(jù)CIAV基因組保守區(qū)域VP2部分基因設(shè)計(jì)合成1對擴(kuò)增片段大小為154bp的特異性引物,同時(shí)通過優(yōu)化反應(yīng)條件建立了快速檢測CIAV的SYBR GreenⅠ熒光定量PCR方法,進(jìn)行敏感性、特異性和重復(fù)性試驗(yàn)。結(jié)果顯示,熒光定量法檢測CIAV的Ct閾值與標(biāo)準(zhǔn)品濃度在6.36×107拷貝/μL-6.36拷貝/μL之間呈現(xiàn)良好的線性關(guān)系,相關(guān)系數(shù)R2=0.999,斜率為-3.186,靈敏度比常規(guī)PCR高1000倍。該方法與禽網(wǎng)狀內(nèi)皮組織增生病病毒(REV)、禽白血病病毒(ALV)、馬立克氏病病毒(MDV)等均無交叉反應(yīng),特異性良好;批內(nèi)和批間重復(fù)試驗(yàn)顯示變異系數(shù)均小于2.5%,呈現(xiàn)良好的可重復(fù)性。在模擬試驗(yàn)中,該方法能夠檢測到1000羽份弱毒疫苗中1個(gè)EID50的低劑量污染,應(yīng)用該方法從送檢的14種(批)商品化禽用弱毒疫苗中檢測到2份為CIAV陽性,陽性樣品按照經(jīng)典的SPF雞檢查法進(jìn)行檢測也為CIAV陽性。綜合上述結(jié)果表明,本研究建立的SYBR GreenⅠ熒光定量PCR檢測方法靈敏度高,特異性強(qiáng),重復(fù)性好,適合用于疫苗中CIAV污染的檢驗(yàn)。二、FAdV PCR檢測方法的建立及其在疫苗污染檢測中的應(yīng)用根據(jù)GeneBank上已發(fā)表FAdV的JSJ13分離株hexon基因,設(shè)計(jì)合成了一對擴(kuò)增片段大小為1310bp的特異性引物,通過優(yōu)化反應(yīng)條件建立PCR檢測方法,并利用該方法對32批弱毒活疫苗進(jìn)行檢測。結(jié)果顯示,其中一支La Sota株的新城疫活疫苗(Clone30)FAdV呈陽性。通過雞胚接種對疫苗中FAdV進(jìn)行分離鑒定,利用建立的PCR法擴(kuò)增,結(jié)果顯示出與疫苗檢測相同的hexon基因條帶,片段長度為1310bp,證明該新城疫活疫苗中存在FAdV污染并對其成功實(shí)現(xiàn)了分離鑒定。本分離株(FAdV-N22)與GenBank上已發(fā)表的其他不同參考株hexon基因的同源性進(jìn)行比較分析并做進(jìn)化樹發(fā)現(xiàn),FAdV-N22株與其他FAdV毒株hexon基因核苷酸序列的同源性介于98.3%-99.8%之間,其中與2015年中國分離株JSJ13株(FAdV-4型)同源性最高,為99.8%;相比之下,與A、B、D和E種同源性較低,與II群和III群的同源性更低,同源性分別僅有56.9%和40.9%;進(jìn)化樹顯示所有毒株分成兩大組,組Ⅰ包含A-E五個(gè)種,組Ⅱ只有屬于FAdV-II亞群的出血性腸炎病毒(HEV)和屬于FAdV-III亞群的產(chǎn)蛋下降綜合征病毒(EDSV)兩個(gè)株,由此證明,FAdV-N22分離株屬于禽腺病毒C種的FAdV-4型。將FAdV-N22分離株進(jìn)行動(dòng)物致病性試驗(yàn),結(jié)果表明,雞接種FAdV-N22后,死亡率為100%,對照組健活。1d齡攻毒后1d全部死亡,無癥狀表現(xiàn);3w齡攻毒后,3d內(nèi)無明顯癥狀,第4d開始出現(xiàn)大量死亡,病雞精神萎靡、羽毛蓬松,肛拭子PCR檢測全部為FAdV陽性。解剖雞出現(xiàn)心包積液等典型臨床癥狀;病理學(xué)組織變化顯示肝臟脂肪變性、出血,肝細(xì)胞核內(nèi)可見嗜堿性包涵體。本研究建立了FAdV的PCR檢測方法,利用這一方法能夠快速有效的檢測弱毒疫苗中FAdV的污染,有利于對弱毒活疫苗中FAdV等家禽外源病毒污染的監(jiān)測。利用此方法并結(jié)合病毒分離鑒定,在我國家禽活疫苗中首次分離鑒定到FAdV-4型,這些結(jié)果表明活疫苗中FAdV的污染可能是FAdV感染雞群并發(fā)病的重要途徑之一。
[Abstract]:Chicken infectious anemia (Chicken infectious, anemia, CIV) is composed of chicken infectious anemia virus (Chicken infectious anemia virus, CIAV) caused by the occurrence of chicken aplastic anemia, with systemic lymphoid tissue atrophy of immune suppression diseases. Avian adenovirus (Fowl adenovirus, FAdV) can cause chickens to produce inclusion B (IBH), pericardial effusion syndrome (HPS) and gizzard erosion (GE) and FAdV.CIAV and other diseases can be caused by infection of chickens in immunosuppressed chickens, the enhancement of susceptibility to other pathogens, at the same time to Marek, the vaccine protection of infectious bursal disease virus and Reticuloendotheliosis disease decreased, resulting in chickens in the total infection or secondary infection of other pathogens such as increase in mortality, causing serious economic losses of.CIAV and FAdV in the vertical and horizontal transmission of SPF and egg production of broiler industry, At the same time can also be spread through contaminated vaccine approaches, this study established a fast detecting method for monitoring CIAV and FAdV vaccine, will help enterprises to quickly and accurately detect avian vaccine in CIAV and FAdV pollution. A Green method for detection of CIAV, SYBR and built in vaccine contamination detection according to the application of CIAV genome conserved regions of VP2 gene were designed and synthesized 1 pairs of specific primers amplified fragment size was 154bp, at the same time through the optimization of reaction conditions for the establishment of a SYBR Green 1 fluorescent quantitative PCR method for rapid detection of CIAV, sensitivity, specificity and repeatability. The results show that the Ct threshold and standard concentration detection the fluorescent quantitative CIAV method in 6.36 * 107 copies / L-6.36 copies / L showed a good linear relationship, the correlation coefficient R2=0.999, the slope is -3.186, sensitivity is 1000 times higher than the conventional PCR. With the method of reticuloendotheliosis virus (REV), avian leukosis virus (ALV), Marek's disease virus (MDV) there were no cross reactions, good specificity; intra and inter repeat test showed that the coefficient of variation was less than 2.5%, showing a good repeatability. In the simulation test, the method able to detect 1000 samples of low dose pollution 1 EID50 attenuated vaccine, from 14 for the application of this method (Group) commercial poultry attenuated vaccine was detected in 2 cases were CIAV positive, the positive samples in accordance with the law of SPF chickens were detected for the inspection of the classic CIAV positive. The above results show that the sensitivity of SYBR Green 1 fluorescent quantitative PCR detection method established in this study, strong specificity, good repeatability, suitable for the inspection of CIAV pollution in the vaccine. In two, the establishment of FAdV method for the detection of PCR and its application in vaccine root pollution detection in GeneBank according to the published FAdV The JSJ13 isolates hexon gene, the amplified fragment size was 1310bp with specific primers were designed and synthesized by optimizing the reaction conditions of the PCR detection method, and to detect the 32 batch of live attenuated vaccine by using this method. The results showed that one La Sota strain of Newcastle disease vaccine (Clone30) was FAdV positive. By isolation and identification of the vaccine in FAdV chicken embryo inoculation, amplified by the PCR method, the results show that the hexon gene with the same vaccine detection zone, the fragment length of 1310bp, the Newcastle disease live vaccine FAdV pollution and its successful implementation. The isolation and identification of isolates (FAdV-N22) were comparative analysis and phylogenetic tree showed that the homology with GenBank has been published on the other reference strains of hexon gene between FAdV-N22 strain and other strains of FAdV hexon gene nucleotide sequence homology between 98.3%-99.8%, which 涓，

本文編號：1534429