1型鸭肝炎病毒R株全基因组分析与检测技术的研究

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

【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6pg·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0.015fg·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。

[Objective] To determine the genomic sequence of a duck hepatitis virus type1 （DHV-1） strain and establish nested PCR and real-time PCR. [Method] Real-time quantitative polymerase chain reaction （RTQ-PCR） assay based on SYBR Green I technology and nested PCR were developed to target 3D gene of DHV-1. [Result] Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV1-R genetic organization is 5＇ UTR-VP0-VP3-VP1- 2A1-2A2-2B-2C-3A-3B-3C-3D-3＇ UTR, DHV1-R has a close realationship with Parechovirus, DHV1-R has 94.2%-99.2% nucleotide sequence identity and has 98%-98.8% amino acid identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using nested PCR and real-time PCR. The results showed that sensitivity of nested PCR is 6pg·ml^-1, and real-time PCR Tm value is 85.6℃, and the detected limit of postive plasmid is 0. 015 fg·μ1^-1. [Conclusion] All results showed that the nested PCR and real-time PCR have high sensitivity and specificity to detect DHV-1. These methods are rapid, sensitive, and reliable, and can be readily adopted for detection of DHV-1 from other clinical samples.