番鸭细小病毒和鸭甲肝病毒3型广西株全基因序列分析及其分子生物学诊断方法的建立

发布时间：2018-06-25 10:28

本文选题：番鸭细小病毒 + 鸭甲肝病毒　；参考：《广西大学》2015年硕士论文

【摘要】：番鸭细小病毒病是由番鸭细小病毒(MDPV)引起的引起的主要感染三周龄以内雏番鸭的一种病毒性传染病,雏鸭感染后可产生肠道卡他性炎症、拉稀腹泻、两脚发软等症状,严重危害了番鸭饲养业的发展。目前,番鸭细小病毒病在我国福建、广东、广西等主要番鸭饲养区广泛流行。鸭甲型病毒性肝炎是由鸭甲肝炎病毒(DHAV)引起的一种传染性强致死率高的传染病,主要侵害三周龄以内雏鸭。DHAV根据血清型分为DHAV-1型、DHAV-2型和DHAV-3型,分别对应于原来的血清Ⅰ型DHV、最早发现于台湾的“新型DHV”和最早发现于韩国的“新型DHV”。随着我国养鸭业的发展,鸭肝炎的发病率和死亡率越来越高,严重制约了养鸭业的发展。近年来,在我国流行的DHAV主要为DHAV-1和DHAV-3。为从分子水平上了解广西流行株MDPV和DHAV-3的遗传变化规律,本研究应用PCR方法扩增了广西分离株MDPV GX2011-5和DHAV-3GXLZ07的不同核酸片段,对其进行了全基因组序列测定与分析。结果表明,GX2011-5全基因序列与GeneBank公布的其他MDPV株的相似性介于90.5%～94.3%之间,与上海SAAS-SHNH株的相似性最高,与匈牙利FM株的相似性为最低；与鹅细小病毒(Goose Parvovirus, GPV)的相似性介于81.4%～91.4%之间,其中与重庆DY株相似性最高,与匈牙利Virulent B株相似性最低。GXLZ07株全基因序列与DHAV-3其它毒株全基因序列相似性介于95.1～99.3%之间,与福建G株相似性最高,与北京B63株相似性最低。根据GenBank中鸭甲肝病毒1型(DHAV-1)的3C基因和DHAV-3的VP0基因分别设计了2对特异性引物,并优化扩增条件,建立了鉴别DHAV-1和DHAV-3二重二温式RT-PCR的检测方法。该方法特异性好,对其他鸭病原体无特异性扩增。该方法的敏感性高,对DHAV-1的核酸最小检测量为4.9g,对DHAV-3的为7.5ng。应用该方法对53份疑似病料进行检测,结果DHAV-1 P日性3份,DHAV-3阳性2份,表明建立的方法适用于临床样品检测的应用。根据GenBank中鸭DHAV-1的3C基因、DHAV-3的VP0基因和MDPV的NS基因分别设计了3对特异性引物,并优化扩增条件,建立了DHAV-1V DHAV-3和MDPV多重PCR的检测方法。该方法特异性好,对其他鸭病原体无特异性扩增。该方法的敏感性高,对DHAV-1的核酸最小检测量为5.2pg,对DHAV-3的为7.9pg,对MDPV的为5.8pg。应用该方法对53份疑似病料进行检测,结果DHAV-1阳性3份,DHAV-3阳性2份,MDPV阳性5份,说明该方法很适用于DHAV-1、DHAV-3和MDPV的鉴别和诊断。
[Abstract]:Muscovy duck parvovirus (MDPV) is a kind of viral infection caused by muscovy duck parvovirus (MDPV), which mainly infects young muscovy ducks within three weeks of age. It seriously endangers the development of muscovy duck breeding. At present, muscovy duck parvovirus is prevalent in Fujian, Guangdong and Guangxi. Duck viral hepatitis A (DHAV) is a highly infectious disease caused by duck hepatitis A virus (DHAV), which mainly infects ducklings within three weeks of age. It is divided into two serotypes: DHAV-1, DHAV-2 and DHAV-3. Corresponding to the original serum type I DHVs, the "new DHV" was first found in Taiwan and the "new-type DHV" in Korea. With the development of duck breeding in China, the morbidity and mortality of duck hepatitis are higher and higher, which seriously restricts the development of duck breeding. In recent years, the popular DHAV in China are DHAV-1 and DHAV-3. In order to understand the genetic variation of MDPV and DHAV-3 in Guangxi at molecular level, different nucleic acid fragments of Guangxi isolates MDPV GX2011-5 and DHAV-3GXLZ07 were amplified by PCR, and their whole genome sequence was determined and analyzed. The results showed that the similarity between GX2011-5 gene sequence and other MDPV strains published by GeneBank ranged from 90.5% to 94.3%, the similarity with Shanghai SAAS-SHNH strain was the highest, and the similarity between GX2011-5 gene sequence and Hungarian FM strain was the lowest. The similarity between GPV and Goose Parvovirus (GPV) was between 81.4% and 91.4%, among which, the similarity with DY strain in Chongqing was the highest, and that with virus B strain in Hungary was the lowest. The total gene sequence of GXLZ07 strain was between 95.1and 99.3% with that of DHAV-3 strain. The similarity was the highest with G strain in Fujian and the lowest with B63 strain in Beijing. Two pairs of specific primers were designed according to the 3C gene of DHAV-1 and the VP0 gene of DHAV-3 in GenBank, and the amplification conditions were optimized. A double temperature RT-PCR method was established for the identification of DHAV-1 and DHAV-3. The method has good specificity and has no specific amplification to other duck pathogens. The sensitivity of this method is high, the minimum detection of DHAV-1 nucleic acid is 4.9 g, and that of DHAV-3 is 7.5 ng. This method was used to detect 53 suspected samples. The results showed that 3 DHAV-1 P samples were positive for DHAV-3, which indicated that the established method was suitable for clinical sample detection. According to DHAV-1 3C gene, DHAV-3 VP0 gene and MDPV NS gene in GenBank, three pairs of specific primers were designed, and the amplification conditions were optimized. A method for the detection of DHAV-1V DHAV-3 and MDPV multiplex PCR was established. The method has good specificity and has no specific amplification to other duck pathogens. The sensitivity of this method is high, the minimum detection of nucleic acid for DHAV-1 is 5.2 PG, for DHAV-3 is 7.9 PG, for MDPV is 5.8 PG. 53 suspected samples were detected by this method. The results showed that 3 DHAV-1 positive and 2 DHAV-3 positive cases were MDPV positive, which indicated that this method was suitable for the differential diagnosis and diagnosis of DHAV-1 and DHAV-3 and MDPV.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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