摘要
为分离鉴定广东某养鸭场疑似"肝坏死"病例的病原,本研究将采集的病料样品感染番鸭胚及番鸭胚成纤维细胞(MDEF)进行病原分离,提取出现细胞病变培养物的核酸,采用宏基因组学方法,以随机引物反转录并合成双链DNA进行PCR扩增,扩增产物进行分子克隆和测序分析。结果显示:F6代分离的病毒滴度为log10^(5.0)TCID_(50)/m L,能够导致9日龄~11日龄番鸭胚死亡及MDEF产生明显细胞病变。50个随机克隆经Gen Bank检索有28个为鸭2型腺病毒(DAd V-2)同源序列,核苷酸同源性为92%~99%。28个同源序列形成8个毗连序列群,共计15 107 bp,覆盖DAd V-2基因组全长的34.54%,分离株与Gen Bank中唯一的DAd V-2 GR分离株亲缘关系最近,其部分hexon基因编码的氨基酸同源性为83.3%。参照测定序列设计的引物能够特异性扩增病毒基因。本研究首次应用宏基因组学分离获得国内首株DAd V-2,也是迄今除了法国以外国内首次报道的DAd V-2分离株。
To identify the pathogen caused the disease with typical hepatitis in Muscovy ducklings, a virus was isolated （in） from duck embryo and Muscovy duck embryo fibroblast （MDEF） inoculated with liver suspension from an affected duckling. The isolate was able to cause duck embryo death and （produce） cytopathic effects （CPE）. By using metagenomic analysis, double- stranded DNA of the virus was synthesized with random primer and the PCR products were cloned for sequencing and analysis. Twenty-eight of fifty randomly selected clones were 92% to 99% homologous to the only sequence of duck adenovirus 2 （DAdV-2） available in the GenBank. Interestingly, the 28 clones were evenly distributed along the entire genome and assembled 8 contigs with a total of 15,107 bp which accounted for 34.54% of the genome. There was 83.3% hexon gene homology of the isolate and the reference DAdV-2 GR strain at the amino acid level. Furthermore, the virus gene was amplified with primers derived from the sequenced clones. This is the first report of the DAdV-2 （named ZLY strain） outside of France, and the first everto use the metagenomic analysis to identify the virus.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2015年第12期903-907,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
广东省产学研项目(2010B090301019)
关键词
宏基因组
鸭2型腺病毒
分离鉴定
metagenomics
duck adenovirus 2
isolation and identification
