中国农业科学 ›› 2008, Vol. ›› Issue (9): 2835-2842 .doi: 10.3864/j.issn.0578-1752.2008.09.036

• 兽医 • 上一篇    下一篇

1型鸭肝炎病毒R株全基因组分析与检测技术的研究

  

  1. 华南农业大学兽医学院
  • 收稿日期:2007-07-11 修回日期:2007-10-01 出版日期:2008-09-01 发布日期:2008-09-01
  • 通讯作者: 张桂红

  1. 华南农业大学兽医学院
  • Received:2007-07-11 Revised:2007-10-01 Online:2008-09-01 Published:2008-09-01

摘要: 【目的】测定1型鸭肝炎病毒(DHV1)毒株R全基因组,建立鸭肝炎病毒1型巢式PCR与实时荧光定量RT-PCR。【方法】设计特异性引物测定DHV1毒株R全基因组,以3D为靶基因序列的引物P1和P2,P3 和P4进行巢式PCR,引物F和R进行实时荧光定量RT-PCR。【结果】序列分析发现该毒株与其它GenBank上发表的DHV1毒株基因组核苷酸序列同源性为94.2%~99.2%,编码聚合蛋白氨基酸序列同源性为98%~98.8%,表明DHV1-R株与其它DHV1毒株之间病毒基因组一级结构有较高的同源性。基因组结构5′UTR-VP0-VP3-VP1-2A1-2A2-2B-2C- 3A-3B-3C-3D-3′UTR在遗传进化关系上与副肠孤病毒属(Parechovirus)亲缘关系较近。参照鸭肝炎病毒1型基因序列设计特异性引物,分别进行巢式PCR和SYBR GreenⅠ实时荧光定量RT-PCR 方法检测鸭肝炎病毒1型, 结果表明巢式PCR敏感性为6 pg•ml-1。实时荧光定量RT-PCR确定特异性产物的Tm值,同时做普通RT-PCR。试验结果表明,特异性产物的Tm值为85.6℃,最低能检测到含0.015 fg•μl-1 阳性质粒标准品。【结论】建立的巢式PCR与SYBR GreenⅠ实时荧光定量RT-PCR 检测方法显示了较好的特异性、敏感性,为鸭肝炎病毒1型的临床检测和流行情况调查提供了新的技术手段。

关键词: 1型鸭肝炎病毒, 全基因组, 巢式PCR, 荧光定量RT-PCR

Abstract: The genome sequence of a duck hepatitis virus type 1 (DHV-1) strain was determined. Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV1-R genetic organaiztion is 5' UTR–VP0–VP3–VP1–2A1–2A2–2B–2C–3A–3B–3C–3D–3' UTR, DHV1-R has colsed realationship with Parechovirus, DHV1-R has 94.2%-99.2% nucleotide sequence homology and has98%-98.8% amino acid homology with other DHV1 strains ,Based on the DHV1 sequence database on Genbank,three pairs of specific primers were designed to amplify duck hepatitis virus type 1 using nested PCR and real-time PCR ,the results showed that sensitivity of nested PCR is 6pg/mL,and real-time PCR Tm value is 85.6℃ ,and the detected limit of postive plasmid is 0. 015fg/μL,all tests showed that nested PCR and real-time PCR have high sensitivity and specificity to detect duck haptitis virus type 1.