禽腺病毒血清4型的分离鉴定及环介导等温扩增技术诊断方法的建立

发布时间：2018-03-09 09:35

  本文选题：禽腺病毒(FAdV)　切入点：分离鉴定　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：禽腺病毒血清4型能引起严重的心包积液综合征,又称“安卡拉”病。2013年以来该病在我国河南、江苏、河北、陕西等地大面积流行,给家禽养殖业造成了巨大的经济损失。本研究从疑似心包积液综合征的发病鸡场采集发病和病死鸡肝脏组织分离到一株病毒。根据GenBank已公布禽腺病毒Hexon基因序列设计特异性PCR检测引物,经PCR检测获得阳性结果。核酸序列比对和遗传进化树分析表明,分离毒株为禽腺病毒血清4型(FAdV-4)。根据GenBank已公布FAdV-4 Hexon基因序列,设计两对LAMP引物,参照LAMP操作指南建立了禽腺病毒血清4型的LAMP检测方法,并与普通PCR检测方法进行了比较研究。试验结果如下:1.采集疑似FAdV-4感染鸡的肝脏,经适当处理后接种9日龄非免疫鸡胚,接种后3~4 d鸡胚开始出现死亡,死亡鸡胚出现胚体卷曲,全身发红,胚体发育不良,明显小于正常同日龄鸡胚。剖检见肝脏肿大出血、表面有灰白色坏死点或呈土黄色。收取感染鸡胚的尿囊液,通过胸肌注射感染健康非免疫的20日龄白羽艾维因肉鸡,感染24 h后开始发病,72 h出现死亡。病死鸡剖检观察到和自然感染一样的病理变化:心包内充盈大量淡黄色液体,肝脏肿大表面有出血点或土黄色坏死斑块。对照组鸡未出现任何临床病理变化,剖检也未见异常。2.依据FAdV-4 Hexon基因序列设计一对PCR检测引物,对含有疑似FAdV-4的鸡胚尿囊液进行PCR检测,回收FAdV阳性PCR检测产物连接载体,测定克隆的部分Hexon基因核酸序列。序列比对结果显示分离毒株与禽腺病毒血清4型江苏株(KU569296.1)同源性高达100%,与火鸡出血性肠炎病毒(AY849321.1)同源性为31.5%、与减蛋综合征病毒(Y09598.1)同源性为30.8%,系统进化树分析显示分离毒株与FAdV-4属同一分支。3.以分离毒株作为FAdV-4阳性模板,以禽腺病毒Hexon基因序列设计两对LAMP检测引物,建立了FAdV-4的LAMP检测方法,扩增产物加入1000×SYBR Green I 1μL,出现黄绿色荧光为FAdV-4阳性,颜色未变化为FAdV-4阴性。该方法的最佳反应温度为61℃,反应时间30 min。与普通PCR检测相比LAMP方法具有更高的灵敏度,检测下限为6.4 fg/μL。对采集的临床发病鸡的21份肝脏样品进行检测,常规PCR检出4份FAdV-4阳性,LAMP方法检出6份FAdV-4阳性,LAMP方法比常规PCR检出率高。
[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，

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