牡蛎疱疹病毒巢式PCR检测法及基因分型系统的建立

发布时间：2018-08-26 07:29

【摘要】：自上世纪90年代开始,包括我国在内的全球多个国家和地区的多种养殖双壳贝类在春夏季水温较高时频繁发生大规模死亡。经流行病学调查和人工感染实验验证,一种疱疹样病毒的感染与这些死亡事件密切相关。国际病毒分类委员会(International Committee on Taxonomy of Virus,ICTV)2012年发布的分类报告将该病毒命名为牡蛎疱疹病毒(Ostreid herpesvirus 1,Os HV-1),该病毒是疱疹病毒目(Herpesviridae)、软体动物疱疹病毒科(Malacoherpesviridae)、牡蛎病毒属(Ostreavirus)下唯一的病毒种。分子流行病学调查显示,与最早发现的病毒株相比,该病毒还有其他病毒株的分化,随着时间推移和空间的转换,该病毒还有新的变异株不断产生并被发现,不同变异株间宿主范围、毒力、发病时间、空间分布等流行病学特征也有所不同。我们的研究目标是建立感染双壳贝类的疱疹病毒主要变异株的检测鉴定方法和基因分型系统,掌握其感染情况,探索其主要变异株的分子流行病学特征,建立健康的养殖模式,做好对该类病害的防控工作。本研究还对引起我国养殖贝类发病的几种寄生虫—派琴虫(Perkinsus spp.)、折光马尔太虫(Marteilia refringens)、包纳米虫(Bonamia spp.)进行了抽样检测,以期掌握我国养殖贝类的寄生虫感染现状,作好针对性的预防和治疗措施,保证贝类养殖业健康、稳定地发展。研究方法、部分结果及结论如下:第一部分:对Os HV-1、AVNV、Os HV-1 SB三个主要变异株的一段保守序列—DNA聚合酶基因(DNA polymerase gene)进行比对,发现三者该区域的序列完全一致。针对这一段序列(约5600bp)利用primer5.0设计巢式PCR的内外引物,外引物的扩增片段长度为573bp,内引物的扩增片段长度为386bp,优化了PCR反应体系和条件,建立了牡蛎疱疹病毒巢式PCR检测法(P-n PCR检测方法)。利用该巢式PCR检测法与基于病毒基因组C区序列建立的巢式PCR检测法(C-n PCR检测方法)对同一批采集于不同年份和宿主来源的Os HV-1疑似感染样本进行检测。结果显示,P-n PCR检测方法能稳定地检出100拷贝/μL的病毒DNA,与C-n PCR检测法相比,具有更强的特异性和更高的检出率。研究表明,本研究建立的P-n PCR检测方法适用于Os HV-1不同变异株的检测,可为该病毒的检测和流行病学调查提供可靠的技术支持。第二部分:利用4对引物C2/C6、Del36-37F2/Del36-37R、IA1/IA2、NC1/NC2对2001年至2014年间采自我国辽宁、山东两省的沿海养殖区的278个贝类病料样本DNA提取物进行PCR扩增,将PCR反应后结果呈Os HV-1阳性的PCR产物送测序公司测序。利用Mega6.0软件分别对每对引物所获得的测序结果,及一些从genebank上下载的Os HV-1主要变异株序列进行比对分析,建立系统发育进化树,研究本实验中样品间及与之前研究中的Os HV-1主要变异株间的序列变化情况及系统发育关系。结果显示,本实验中所选的278个样品中的Os HV-1变异株,在基因组的C2/C6区、NC1/NC2区发生了较大的变异,其中根据C2/C6区序列的变化118个样本产生了11个变异株,根据NC1/NC2区序列的变化56个样本产生了26个变异株。这些变异株除了在C2/C6区的微卫星区产生较大的变异外,其余变化均为零星碱基的增添、缺失或改变。综合4个区域的碱基变化情况,最终确定实验中所选的所有Os HV-1变异株与AVNV和Os HV-1 SB关系较近,与Os HV-1和Os HV-1μVar关系较远,与之前研究中的病毒流行病学特征相符。本实验对全面研究感染我国双壳贝类的Os HV-1的基因分型和系统进化关系提供了借鉴。第三部分:采用世界动物卫生组织(OIE)推荐的检测派琴虫(Perkinsus spp.)、折光马尔太虫(Marteilia refringens)、包纳米虫(Bonamia spp.)的引物对实验室保存的2007-2013年间采集于我国辽宁、山东、广东、广西、福建等省份及韩国沿海地区的牡蛎、贻贝、蛤蜊、扇贝等进行PCR检测。结果显示,派琴虫的感染主要发生在辽宁大连、山东青岛、山东荣成、山东潍坊,其他地区未发现该原虫的感染。从发生感染的时间上看,主要发生在7、8、9月水温相对较高的季节。从宿主的种类上看,受感染的宿主主要为贻贝、菲律宾蛤仔和毛蚶。此次检测中未发现折光马尔太虫和包纳米虫的感染。
[Abstract]:Since the 1990s, large-scale deaths of bivalve shellfish have occurred frequently in many countries and regions around the world, including China, in spring and summer when the water temperature is high. Epidemiological investigation and artificial infection experiments have proved that a herpes-like virus infection is closely related to these deaths. International Virus Classification Committee (I) The taxonomic report of the International Committee on Taxonomy of Virus (ICTV) published in 2012 named the virus Ostreid herpes virus 1 (Os HV-1), which is the only virus species under Herpes viridae, Malacoherpesviridae and Ostreavirus. The epidemiological investigation showed that there were other strains of the virus differentiated from the earliest one. With the passage of time and the transformation of space, new variants of the virus were produced and found continuously. The epidemiological characteristics of host range, virulence, onset time and spatial distribution among different variants were also different. The aim of this study is to establish a method for detection and identification of the main variants of herpesvirus infecting bivalve shellfish and a genotyping system, to master the infection situation, to explore the molecular epidemiological characteristics of the main variants, to establish a healthy breeding model, and to do a good job in the prevention and control of such diseases. Several kinds of parasites, Perkinsus spp., Marteilia refringens and Bonamia spp., were sampled and tested in order to know the status of parasite infection in shellfish farming in China, and to take preventive and therapeutic measures to ensure the healthy and stable development of shellfish farming industry. The results are as follows: Part 1: DNA polymerase gene, a conserved sequence of Os HV-1, AVNV and Os HV-1 SB, was compared and found to be identical in this region. For this sequence (about 5600 bp), primer 5.0 was used to design the internal and external primers of nested PCR and amplified fragments of external primers. The length of the amplified fragment was 573 bp, and the length of the amplified fragment was 386 bp. The PCR reaction system and conditions were optimized. A nested PCR assay for Oyster Herpesvirus (P-n PCR) was established. The results showed that the P-n PCR method could detect 100 copies/mu L of virus DNA stably. Compared with the C-n PCR method, it had stronger specificity and higher detection rate. To provide reliable technical support for the detection and epidemiological investigation of the virus. Part II: DNA extracts from 278 shellfish samples collected from Liaoning and Shandong coastal culture areas from 2001 to 2014 were amplified by PCR using four pairs of primers C2/C6, Del36-37F2/Del36-37R, IA1/IA2 and NC1/NC2, and the PCR results were presented as Os HV-1. The positive PCR products were sent to the sequencing company for sequencing. The sequencing results obtained by each pair of primers and the sequences of some major Os HV-1 mutants downloaded from genebank were analyzed by Mega 6.0 software respectively. A phylogenetic tree was established to study the sequence between samples and the major Os HV-1 mutants previously studied. The results showed that in the C2/C6 region of the genome, there were significant variations in the NC1/NC2 region of the Os HV-1 mutant strain from 278 samples selected in this study. Among them, 11 mutant strains were produced from 118 samples according to the variation of C2/C6 region sequence, and 26 mutant strains were produced from 56 samples according to the variation of NC1/NC2 region sequence. Except for the large variation in the microsatellite region of C2/C6 region, the other changes were sporadic base addition, deletion or alteration. Based on the analysis of the base changes in the four regions, it was determined that all the Os HV-1 mutants selected in the experiment had close relationship with AVNV and Os HV-1SB, and had far relationship with Os HV-1 and Os HV-1 mu Var. The results of this study are consistent with the epidemiological characteristics of the viruses. This study provides a reference for the comprehensive study of the genotyping and phylogenetic relationship of Os HV-1 in bivalve shellfish infected in China. Part III: The detection of Perkinsus spp., Marteilia refringens, and Bon nanoparticles recommended by the World Organization for Animal Health (OIE) The primers of AMIA spp. were collected from Liaoning, Shandong, Guangdong, Guangxi, Fujian provinces and coastal areas of South Korea from 2007 to 2013. The results showed that the infection of Parthenocissus spp. mainly occurred in Dalian, Shandong, Qingdao, Rongcheng, Weifang, Shandong and other areas. The infection occurred mainly in the relatively high water temperature seasons of July, August and September. The host species were mainly mussels, clams and clams. No refractive Maltesia and hydatid infection was found in this test.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S944

【相似文献】