發(fā)布時(shí)間：2018-03-09 09:35

  本文選題：禽腺病毒(FAdV)　切入點(diǎn)：分離鑒定　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：禽腺病毒血清4型能引起嚴(yán)重的心包積液綜合征,又稱“安卡拉”病。2013年以來該病在我國河南、江蘇、河北、陜西等地大面積流行,給家禽養(yǎng)殖業(yè)造成了巨大的經(jīng)濟(jì)損失。本研究從疑似心包積液綜合征的發(fā)病雞場采集發(fā)病和病死雞肝臟組織分離到一株病毒。根據(jù)GenBank已公布禽腺病毒Hexon基因序列設(shè)計(jì)特異性PCR檢測引物,經(jīng)PCR檢測獲得陽性結(jié)果。核酸序列比對和遺傳進(jìn)化樹分析表明,分離毒株為禽腺病毒血清4型(FAdV-4)。根據(jù)GenBank已公布FAdV-4 Hexon基因序列,設(shè)計(jì)兩對LAMP引物,參照LAMP操作指南建立了禽腺病毒血清4型的LAMP檢測方法,并與普通PCR檢測方法進(jìn)行了比較研究。試驗(yàn)結(jié)果如下:1.采集疑似FAdV-4感染雞的肝臟,經(jīng)適當(dāng)處理后接種9日齡非免疫雞胚,接種后3~4 d雞胚開始出現(xiàn)死亡,死亡雞胚出現(xiàn)胚體卷曲,全身發(fā)紅,胚體發(fā)育不良,明顯小于正常同日齡雞胚。剖檢見肝臟腫大出血、表面有灰白色壞死點(diǎn)或呈土黃色。收取感染雞胚的尿囊液,通過胸肌注射感染健康非免疫的20日齡白羽艾維因肉雞,感染24 h后開始發(fā)病,72 h出現(xiàn)死亡。病死雞剖檢觀察到和自然感染一樣的病理變化:心包內(nèi)充盈大量淡黃色液體,肝臟腫大表面有出血點(diǎn)或土黃色壞死斑塊。對照組雞未出現(xiàn)任何臨床病理變化,剖檢也未見異常。2.依據(jù)FAdV-4 Hexon基因序列設(shè)計(jì)一對PCR檢測引物,對含有疑似FAdV-4的雞胚尿囊液進(jìn)行PCR檢測,回收FAdV陽性PCR檢測產(chǎn)物連接載體,測定克隆的部分Hexon基因核酸序列。序列比對結(jié)果顯示分離毒株與禽腺病毒血清4型江蘇株(KU569296.1)同源性高達(dá)100%,與火雞出血性腸炎病毒(AY849321.1)同源性為31.5%、與減蛋綜合征病毒(Y09598.1)同源性為30.8%,系統(tǒng)進(jìn)化樹分析顯示分離毒株與FAdV-4屬同一分支。3.以分離毒株作為FAdV-4陽性模板,以禽腺病毒Hexon基因序列設(shè)計(jì)兩對LAMP檢測引物,建立了FAdV-4的LAMP檢測方法,擴(kuò)增產(chǎn)物加入1000×SYBR Green I 1μL,出現(xiàn)黃綠色熒光為FAdV-4陽性,顏色未變化為FAdV-4陰性。該方法的最佳反應(yīng)溫度為61℃,反應(yīng)時(shí)間30 min。與普通PCR檢測相比LAMP方法具有更高的靈敏度,檢測下限為6.4 fg/μL。對采集的臨床發(fā)病雞的21份肝臟樣品進(jìn)行檢測,常規(guī)PCR檢出4份FAdV-4陽性,LAMP方法檢出6份FAdV-4陽性,LAMP方法比常規(guī)PCR檢出率高。
[Abstract]:Avian adenovirus serotype 4 can cause severe pericardial effusion syndrome, also known as "Ankara" disease. Since 2013, the disease has been widespread in Henan, Jiangsu, Hebei and Shaanxi provinces in China. In this study, a virus was isolated from the liver tissues of chickens with suspected pericardial effusion syndrome. The Hexon gene sequence of avian adenovirus has been published according to GenBank. Specific PCR detection primers were designed. Positive results were obtained by PCR detection. Nucleic acid sequence alignment and genetic phylogenetic tree analysis showed that the virus strain was isolated from avian adenovirus serotype 4 FAdV-4. According to the published sequence of FAdV-4 Hexon gene by GenBank, two pairs of LAMP primers were designed. A LAMP detection method for avian adenovirus serotype 4 was established according to the LAMP operating guidelines, and compared with the conventional PCR detection method. The results were as follows: 1. The liver of chickens suspected to be infected with FAdV-4 was collected. After proper treatment, 9 days old non-immunized chicken embryos were inoculated, and 3 ~ 4 days after inoculation, the chicken embryos began to die, the dead chicken embryos appeared curly embryo body, the whole body became red, the embryo body developed dysplasia, which was obviously smaller than that of the normal chicken embryo of the same day age. The liver swelling and bleeding were observed in the dissection. Grayish-white spots of necrosis or yellowish brown on the surface. Collect allantoic fluid from infected chicken embryos and inject healthy, non-immunized, 20-day-old white feather Evien broilers by intramuscular injection. Death occurred at 72 hours after 24 hours of infection. The pathological changes of dead chickens were the same as those of natural infection: the pericardium was filled with a large amount of yellowish fluid. There were haemorrhage spots or khaki-yellow necrotic plaques on the hepatomegaly surface. There were no clinicopathological changes in the control group and no abnormal .2. according to the sequence of FAdV-4 Hexon gene, a pair of PCR primers were designed. The chicken embryo allantoic fluid containing suspected FAdV-4 was detected by PCR, and the ligation vector of FAdV positive PCR detection product was recovered. The nucleic acid sequence of partial Hexon gene cloned was determined. The sequence alignment results showed that the isolated virus had a high homology with avian adenovirus serotype 4 Jiangsu strain KU569296.1), and had a high homology with turkey hemorrhagic enteritis virus (AY849321.1), and with egg drop syndrome virus (AY849321.1). The phylogenetic tree analysis showed that the isolated strain was the same branch of FAdV-4. The isolated strain was used as FAdV-4 positive template. Two pairs of LAMP primers were designed based on the sequence of avian adenovirus Hexon gene, and the LAMP detection method of FAdV-4 was established. The amplification product was added 1 000 脳 SYBR Green I 1 渭 L, the yellow green fluorescence was FAdV-4 positive and the color did not change to FAdV-4 negative. The optimum reaction temperature of the method was 61 鈩，

本文編號：1587966