兔出血症病毒遗传变异分析及RT-PCR检测的方法建立

范文编号:SW001  范文字数:8715,页数:19

摘 要：目的  获得兔出血症病毒（浙江分离株）近20年间遗传变异概况，建立兔出血症病毒的RT—PCR检测方法。方法  对1989-2006年间的7株RHDV浙江分离株的衣壳蛋白基因（VP60）进行了克隆与测序，并与国内、外RHDV的基因组序列进行了遗传比对和分析；根据兔出血症病毒株的VP60设计引物，对20只人工感染兔的实质脏器、全血和350只实验兔全血样本进行了检测。结果  7株RHDV的VP60基因均由1740个核苷酸组成，编码580个氨基酸。它们与参考序列之间的氨基酸同源性为94.0% ～ 99.8%。系统发生树分析结果显示，RHDV可划分为2个大的基因群，浙江（中国）的RHDV分离株主要集中于C亚群。RT—PCR检测方法表明20只实验兔全部扩出预期条带，而350只实验兔全血检测中出现13只RHDV可疑样本。经血凝抑制试验检测，13例PCR阳性样品中有10个HAI阳性，3个阴性。检测敏感度达100%，特异性为99.12%, 经统计检验，kappa=0.865，表明PCR检出RHDV的结果与HAI高度一致。结论 RHDV基因群之间的遗传距离有逐渐加大的趋势。我们建立的RT—PCR法可用于RHDV浙江分离株的检测，用RT—PCR检测全血中RHDV方法的建立为活体检测RHDV打下了基础。
关键词：兔病毒性出血症病毒；衣壳蛋白基因；遗传变异；RT—PCR
 
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本范文研究内容 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 

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