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当前位置: 首页 > 文章 > 1型鸭肝炎病毒R株全基因组分析与检测技术的研究 中国农业科学 2008,41 (9) 2835-2842
Position: Home > Articles > Genomic Analysis and Establishment of Diagnostic Technology for Duck Hepatitis Virus Type 1 Scientia Agricultura Sinica 2008,41 (9) 2835-2842

1型鸭肝炎病毒R株全基因组分析与检测技术的研究

作  者：
罗玉均;张桂红;陈建红;廖明;徐小芹
单  位：
华南农业大学兽医学院;佛山科学技术学院动物医学系
关键词：
1型鸭肝炎病毒;全基因组;巢式PCR;荧光定量RT-PCR
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。
译  名：
Genomic Analysis and Establishment of Diagnostic Technology for Duck Hepatitis Virus Type 1
作  者：
LUO Yu-jun1,2, ZHANG Gui-hong1, CHEN Jian-hong2, LIAO Ming 1, XU Xiao-qin1, REN Tao1, ZHANG Ji-pei2, LUO Kai-jian1 (1College of Veterinary Medicine, South China Agricultural University, Guangdong 510642; 2College of Life and Science, Foshan University, Nanhai 528231, Guangdong)
关键词：
Duck hepatitis virus type 1; Complete genome; Nested PCR; Real-time RT-PCR
摘  要：
【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·μl-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.

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