The Genetic and Variation Characteristics of VP60 Gene in Rabbit Hemorrhagic Disease Virus: a Analysis Based on RT-PCR Technique
Abstract: Objective To investigate the genetic and variation characteristics of VP60 gene in rabbit hemorrhagic disease virus isolated from Zhejiang, China during last two decades. Method Seven ZJ isolates (RHDV) were included in the study. The VP60 gene of them were cloned, sequenced and blasted with the sequences submitted to GenBank by other authors. After that, a fitness test was performed to verify the RT-PCR protocol based on samples from 20 rabbits artificially infected with RHDV isolates by VP60 primers. Results The sequenced results revealed that the VP60 gene was 1740 bp and encoded a protein of 580 amino acids. The identity of the seven tested Zhejiang RHDV isolates ranged from 94.0% to 99.8%. A phylogenetic analysis presented the VP60 gene was classified into two groups and the Zhejiang RHDV isolates was mainly classified into group I of RHDV genes. The fitness test presented that the RT-PCR protocol established could distinguish out all of the 20 RHDV artificially infected rabbits. The results of field test showed that only 13 rabbits were turned out to be RHDV positive from 350 animals from different regions of Zhejiang province. There are 10 samples in the 13 PCR-positive samples presented HAI in HAI test, A kappa tests was performed to compare the fitness of the results from PCR with the results of HAI. The results revealed that the PCR diagnosis of RHDV presented high consistency in comparison with HAI (kappa = 0.865, p<0.01) i.e. PCR method has the same discriminating ability to identify the RHDV from rabbit samples. Conclusion The RT-PCR protocol developed by this paper is a stable and suitable method in the detection of RHDV, the results also suggested that there was a increasing trend in the genetic distance of the VP60 gene in RHDV。
Keywords:Rabbit hemorrhagic disease virus;Vp60;Genetic variable; RT-PCR
目 录
中文摘要 I
英文摘要 II
目录 III
1. 绪论 1
1.1 兔出血症简介 1
1.1.1兔出血症的危害及特点 1
1.2兔出血症病毒(RHDV)的形态特征和基因组结构 1
1.3 实验动物兔出血症病毒(RHDV)的防治和检测方法 2
1.4本论文研究内容 4
2. 实验部分 5
2.1 材料 5
2.2 实验方法 5
3. 结果与讨论 9
3.1 RHDV核衣壳蛋白基因的克隆和鉴定 9
3.2分离的6株RHDV的核衣壳蛋白基因与国内外其它毒株的同源性分析 9
3.3 RHDV核衣壳蛋白基因的进化树分析 10
3.4 RT—PCR方法与检测结果 12
3.5 讨论 13
4.总结与展望 16
4.1 实验总结 16
4.1 实验展望 16
致谢 17
参考文献 19