兔出血癥病毒遺傳變異分析及RT-PCR檢測的方法建立

生物工程論文編號:SW001  論文字數:8715,頁數:19

目  錄
中文摘要 I
英文摘要 II
目錄 III
1. 緒論 1
 1.1 兔出血癥簡介 1
 1.1.1兔出血癥的危害及特點 1
 1.2兔出血癥病毒（RHDV）的形態特征和基因組結構 1
 1.3 實驗動物兔出血癥病毒（RHDV）的防治和檢測方法 2 
 1.4本論文研究內容 4
2. 實驗部分 5
 2.1 材料 5
 2.2 實驗方法 5
3. 結果與討論 9
 3.1  RHDV核衣殼蛋白基因的克隆和鑒定 9
 3.2分離的6株RHDV的核衣殼蛋白基因與國內外其它毒株的同源性分析 9
 3.3  RHDV核衣殼蛋白基因的進化樹分析 10
 3.4  RT—PCR方法與檢測結果 12
 3.5  討論 13
4．總結與展望 16
 4.1  實驗總結 16
 4.1  實驗展望 16
致謝 17
參考文獻 19 

摘 要：目的  獲得兔出血癥病毒（浙江分離株）近20年間遺傳變異概況，建立兔出血癥病毒的RT—PCR檢測方法。方法  對1989-2006年間的7株RHDV浙江分離株的衣殼蛋白基因（VP60）進行了克隆與測序，并與國內、外RHDV的基因組序列進行了遺傳比對和分析；根據兔出血癥病毒株的VP60設計引物，對20只人工感染兔的實質臟器、全血和350只實驗兔全血樣本進行了檢測。結果  7株RHDV的VP60基因均由1740個核苷酸組成，編碼580個氨基酸。它們與參考序列之間的氨基酸同源性為94.0% ～ 99.8%。系統發生樹分析結果顯示，RHDV可劃分為2個大的基因群，浙江（中國）的RHDV分離株主要集中于C亞群。RT—PCR檢測方法表明20只實驗兔全部擴出預期條帶，而350只實驗兔全血檢測中出現13只RHDV可疑樣本。經血凝抑制試驗檢測，13例PCR陽性樣品中有10個HAI陽性，3個陰性。檢測敏感度達100%，特異性為99.12%, 經統計檢驗，kappa=0.865，表明PCR檢出RHDV的結果與HAI高度一致。結論 RHDV基因群之間的遺傳距離有逐漸加大的趨勢。我們建立的RT—PCR法可用于RHDV浙江分離株的檢測，用RT—PCR檢測全血中RHDV方法的建立為活體檢測RHDV打下了基礎。
關鍵詞：兔病毒性出血癥病毒；衣殼蛋白基因；遺傳變異；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