1型鸭肝炎病毒R株全基因组分析与检测技术的研究

 【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。

Genomic Analysis and Establishment of Diagnostic Technology for Duck Hepatitis Virus Type 1

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.