烟草环斑病毒和番茄环斑病毒的半巢式RT-PCR检测

烟草环斑病毒(Tobacco ringspot virus,TRSV)和番茄环斑病毒(Tomato ringspot virus,ToRSV)是我国禁止入境的检疫性有害生物。采用半巢式RT-PCR方法对这两种病毒进行了检测,用Trizol快速提取病毒总RNA,并根据TRSV和ToRSV的外壳蛋白基因设计特异性引物,经过第一轮RT- PCR和第二轮半巢式PCR扩增,TRSV样本分别得到359 bp和206 bp特异性片段,ToRSV样本分别得到340 bp和219 bp特异性片段。半巢式RT-PCR扩增产物的测序表明,TRSV产物序列与GenBank中登录的外壳蛋白基因存在88%～97%的同源性,ToRSV产物序列与GenBank中登录的外壳蛋白基因存在96%～100%的同源性。研究显示,DAS-ELISA与RT-PCR的检测灵敏度相近,而半巢式RT-PCR的检测灵敏度比这两种方法高出10~3倍以上。     

齿兰环斑病毒与建兰花叶病毒分子检测研究

齿兰环斑病毒（Odontoglossum ringspot virus,ORSV）与建兰花叶病毒（Cymbidium mosaic virus, CyMV）是严重危害兰科植物的两种主要病毒。本研究根据病毒外壳蛋白基因设计特异性引物,应用ELISA、普通RT-PCR、巢式RT-PCR和免疫捕获RT-PCR4种方法进行了检测研究与比较。结果表明：普通RT-PCR与ELISA方法检测灵敏度相当;巢式RT-PCR检测灵敏度要比普通RT-PCR与ELISA方法高出104倍以上;免疫捕获RT-PCR检测灵敏度介于普通RT-PCR和巢式RT-PCR之间。采用巢式RT-PCR方法对我国台湾进境的蝴蝶兰植株样本检测,1号样本出现与阳性对照一致的特异条带。双向测序分析,扩增产物序列与ORSV外壳蛋白基因具有100％的同源性,表明1号蝴蝶兰样本携带ORSV。     

北京怀桑地区李矮缩病毒的检测鉴定

在北京怀柔地区核果类果树病毒疫情调查中,对樱桃树上有矮缩、斑驳、皱缩等症状的叶片样品,通过酶联免疫检测为阳性反应,进行RT-PCR鉴定、外壳蛋白基因扩增片段测序,发现该序列与李属坏死环斑病毒(PDV)ch株系分离物(L28145.1)序列的同源性为100%,与分离物(AF208746.1,AF208747.1,AF208743.1)序列的同源性大干97%,说明被检测样品感染有PDV.这是PDV病毒在北京地区发生的首次报道,对该病毒来源及其流行趋势也进行了分析.     

进境玫瑰鲜切花李属坏死环斑病毒的检测鉴定

近期上海口岸连续多次从进境的玫瑰鲜切花中检出我国检疫性有害生物李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),通过DAS-ELISA、RT-PCR、SYBR Green I实时荧光RT-PCR和序列分析等4种方法分别检测和复验该病毒,结果均为阳性,由此确定从进境玫瑰鲜切花中检出了PNRSV。同时本文分别检测玫瑰叶片、花瓣、茎干和花萼,结果均带有PNRSV,确定该病毒系统侵染玫瑰。     

从进境的蝴蝶兰上检出齿兰环斑病毒

齿兰环斑病毒 (OdontoglossumringspotvirusORSV)是兰花上的主要病毒 ,可侵染多种兰科植物 ,在卡特兰和建兰上危害尤为严重。在蝴蝶兰上 ,叶表现轻花叶 ,花畸形或发育不良 ,严重影响花的数量和品质。 2 0 0 2年9月 ,我们从来自台湾的蝴蝶兰中发现可疑症状植株 ,经透射电镜观察和血清学检测为齿兰环斑病毒。1　材料和方法1 1　材料进境的蝴蝶兰苗 ,花叶坏死症状 ( 1号样品 )、没有症状的 ( 2号样品 )取叶片进行检测 ;2 %的磷钨酸 (厦门大学化学系提供 ) ;血清学试剂和阳性对照由国家质检总局动物检所提供 ;本实验室保存的无病毒蝴蝶兰植株…     

半巢式RT-PCR检测进口大豆中菜豆荚斑驳病毒的研究

 菜豆荚斑驳病毒(Bean pod mottle virus,BPMV)在美国东部和南部的大豆产区广泛分布。该病毒能够造成3%~52%的产量损失并使大豆品质降低,目前我国未见其分布。针对进口大豆种子的植物病毒检测,本文建立了半巢式RT-PCR技术检测BPMV的方法。该方法采用Trizol快速提取大豆种子病毒总RNA,并根据BPMV的外壳蛋白编码基因设计特异性引物,经第一轮RT-PCR和第二轮半巢式PCR扩增,分别得到275 bp和196 bp大小的特异性基因片段。半巢式RT-PCR扩增产物的测序表明,该产物序列与BPMV的外壳蛋白编码基因存在91%~95%的高度同源性。检测灵敏度比较研究显示,DAS-ELISA与RT-PCR的检测灵敏度相近,半巢式RT-PCR的检测灵敏度比这2种方法高出103倍以上。     

两种主要甜樱桃病毒RT-PCR检测方法的改进

李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)和李矮缩病毒(Prune dwarf virus,PDV)是较为常见且危害严重的两种植物病毒,1992年被我国列为进境检疫危险性有害生物,主要危害李属和蔷薇属植物.反转录-聚合酶链式反应检测法(RT-PCR)因操作简便且灵敏度高而备受青睐.利用该方法已分别从北京怀柔[1-2]及大连地区[3]栽培的桃、樱桃中检测到PNRSV和PDV两种病毒.由于PNRSV和PDV同属雀麦花叶病毒科等轴不稳环斑病毒属,且传播方式相似,往往同时发病.已有的RT-PCR检测方法,一次反转录操作仅能检测一种病毒,耗时长、效率低,急需建立新的RT-PCR检测体系.本试验采用随机六聚体引物进行反转录,对RT-PCR检测方法进行了改进.     

用毕赤酵母表达李属坏死环斑病毒外壳蛋白基因及表达产物抗血清的制备与研究

用RT-PCR的方法克隆李属坏死环斑病毒的外壳蛋白(CP)基因,然后将其连接到pGEM-T载体上,得到pGEM-T-PNRSV重组载体,然后将其克隆到真核表达载体pPIC9K上,转化毕赤酵母GS115菌株后,在甲醇的诱导下表达外壳蛋白,表达产物经过SDS-PAGE及Western-blot分析,结果表明该病毒的CP基因在毕赤酵母中得到了高效表达,回收表达蛋白并免疫家兔,获得PNRSV特异性抗血清共计40mL,经过间接酶联免疫吸附法测定其效价为3.2×104,本研究利用基因克隆的方法来制备PNRSV抗血清,完全避免了用该病毒的繁殖来制备抗血清而导致该病毒扩散蔓延的风险。     

四个李属坏死环斑病毒分离物的检测鉴定及CP基因序列分析

采用RT-PCR方法,从北京昌平、云南昆明、陕西西安和浙江海宁的樱桃和月季罹病叶片中检测到李属坏死环斑病毒,为分析我国李属坏死环斑病毒分子生态学特性,克隆了病毒分离物的CP基因并进行序列分析。结果显示,北京分离物的CP基因长675nt、编码224aa,而西安、昆明和海宁分离物的CP基因均为681nt、编码226aa。序列相似性分析表明,4个分离物的CP基因之间具 有很高的同源性, 各个分离物间的核苷酸同源性在94.8%~99.3%之间, 氨基酸同源性在94.2%~98.7%之间。序列比对与系统发生树的分析结果显示,北京分离物属于GroupⅡ组,昆明、西安和海宁分离物属于Group Ⅰ组。     

我国部分地区樱桃病毒病害初步调查和病原检测

对山东泰安、辽宁大连和北京的樱桃病毒病发生情况进行调查,发现8个果园/栽培区均有病毒病发生,主要症状为叶片皱缩、畸形、卷叶、花叶、植株矮缩等。采集20份样品,利用12种病毒的引物进行RT-PCR检测。结果表明,在样品中扩增出与樱桃病毒A(Cherry virus A,CVA)、李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),李矮缩病毒(Prune dwarf virus,PDV)、李树皮坏死与茎痘伴随病毒(Plum bark necrosis stem pitting-associated virus,PBNSPaV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃小果病毒-1(Little cherry virus-1,LChV-1)预期大小一致的目的片段;序列分析表明,与GenBank中注册所测的病毒核苷酸序列均具有较高的一致性。其中,大连、泰安和北京样品均检测到CVA;大连和北京样品中检测到PNRSV和PDV;北京样品中检测到PBNSPaV;大连苗木样品枝条中检测到CGRMV和LChV-1。这是在我国樱桃上首次检测到LChV-1。     

李属坏死环斑病毒怀柔分离物CP基因的克隆、原核表达及其抗血清制备

本研究通过RT-PCR技术获得了包含李属坏死环斑病毒怀柔分离物CP蛋白基因的DNA片段,构建了针对pET29a载体的两种表达质粒;〖JP2〗转化大肠杆菌BL21(DE3),并经IPTG诱导表达了带不同融合肽段的PNRSV1（25 ku）〖JP〗和PNRSV2（29 ku）融合蛋白。经过Ni NTA亲和柱与SDS PAGE分离纯化,获得大量表达融合蛋白,并免疫家兔制备了融合表达蛋白的特异性抗体。间接ELISA测定其效价分别为8×103和3.2×104。     

北京月季病原病毒的高通量测序鉴定和RT-PCR检测

 本研究利用高通量测序技术对北京地区的月季染病样品进行了病毒鉴定,通过序列比对和拼接获得了李属坏死环斑病毒(prunus necrotic ringspot virus,PNRSV)、苹果茎沟病毒(apple stem grooving virus,ASGV)、柑橘碎叶病毒(citrus tatter leaf virus,CTLV)、月季黄叶病毒(rose yellow leaf virus,RYLV)、月季黄花叶病毒(rose yellow mosaic virus,RoYMV)、月季潜隐病毒1(rose cryptic virus1,RCV1)和玫瑰黄脉病毒(rose yellow vein virus,RYVV)等7种已知病毒和2种未知病毒的序列信息。分别对PNRSV,ASGV和RYLV园博园分离物的外壳蛋白基因进行系统进化分析,结果表明PNRSV-YBY归属于PV32组,ASGV-YBY归属于Ⅰ组,RYLV-YBY为玫瑰叶畸形病毒(Rosa rugosa leaf distortion virus, RrLDV)的一个新的分离物。对中国农业大学校园、北京植物园、北京动物园和北京园博园的37份样品进行了PNRSV、ASGV和RYLV的RT-PCR检测,检出率分别为41.7%、44.4%和10.8%。本研究初步明确了侵染北京月季的病毒种类和侵染情况,为月季病毒病的检测和防控提供参考。     

Survey of Prunus necrotic ringspot virus in Rose and Its Variability in Rose and Prunus spp

ABSTRACT A survey for viruses in rose propagated in Europe resulted in detection of only Prunus necrotic ringspot virus (PNRSV) among seven viruses screened. Four percent of cut-flower roses from different sources were infected with PNRSV. Progression of the disease under greenhouse conditions was very slow, which should make this virus easy to eradicate through sanitary selection. Comparison of the partial coat protein gene sequences for three representative rose isolates indicated that they do not form a distinct phylogenetic group and show close relations to Prunus spp. isolates. However, a comparison of the reactivity of monoclonal antibodies raised against these isolates showed that the most prevalent PNRSV serotype in rose was different from the most prevalent serotype in Prunus spp. All of the 27 rose isolates tested infected P. persica seedlings, whereas three of the four PNRSV isolates tested from Prunus spp. were poorly infectious in Rosa indica plants. These data suggest adaptation of PNRSV isolates from Prunus spp., but not from rose, to their host plants. The test methodologies developed here to evaluate PNRSV pathogenicity in Prunus spp. and rose could also help to screen for resistant genotypes.     

我国梅树上病毒及类病毒的检测和鉴定

目前, 我国梅树上的病毒种类及发生情况仍不完全清楚。本研究从北京、武汉、南京和无锡的梅园中采集了64份疑似感染病毒的叶片样品, 通过RT-PCR和斑点杂交, 对7种病毒和2种类病毒进行了检测。共检测到6种病毒和1种类病毒。其中, 李属坏死环斑病毒(prunus necrotic ringspot virus, PNRSV)和桃潜隐花叶类病毒(peach latent mosaic viroid, PLMVd)为我国梅树上的首次检出。PNRSV、亚洲李属病毒2号(Asian prunus virus 2, APV2)、桃叶痘伴随病毒(peach leaf pitting-associated virus, PLPaV)的检出率高于30%。综合考虑病毒的分布及检出率, PLPaV、APV2、PNRSV和李树皮坏死茎痘伴随病毒(plum bark necrosis stem pitting-associated virus, PBNSPaV)是武汉、南京和无锡梅树上的主要病毒。此外, 通过克隆和测序, 获得了PLMVd和梅树病毒A(mume virus A, MuVA)的基因组, PLPaV的RNA1组分和PNRSV外壳蛋白(CP)基因序列。序列比较分析显示, 我国PLMVd梅分离物和PNRSV梅分离物与我国桃分离物亲缘关系最近, 表明PLMVd和PNRSV可能在梅和桃树间交互侵染;我国MuVA梅分离物序列与日本梅分离物序列的相似性高达98.56%;PLPaV梅分离物与我国桃分离物之间序列变异较大。上述结果不仅进一步明确了我国梅树上的病毒及类病毒种类和分布情况, 而且有助于深入了解它们的流行与传播。     

侵染月季的李坏死环斑病毒的检测及CP基因序列分析

近年来，随着我国切花月季（Rosa hybrid）种植面积的不断增加，月季病毒病的发生日趋严重。据笔者调查，平均发病率20％～30％，有的品种如“绿宝石”，发病率超过60％。李坏死环斑病毒（Prunus necrotic ring spot virus，PNRSV）是月季的主要病毒之一。国内对月季病毒病的研究较少。为明确我国切花月季的主要病毒种类，加强对月季种苗质量的监督检测，笔者对侵染月季的PNRSV云南分离物（PNRSV—yunnan）进行了CP基因克隆及序列分析，建立了快速RT—PCR检测技术。     

侵染肥城桃的病毒和类病毒的分子检测与鉴定

为明确山东肥城桃种植区桃树上主要存在的病毒和类病毒及其发生情况,采集具有花叶、斑驳和皱缩典型症状的肥城桃样品,提取叶片总RNA后,分别选用桃树上已报道的啤酒花矮化类病毒Hopstuntviroid(HSVd)、桃潜隐花叶类病毒Peach latent mosaic viroid(PLMVd)、苹果褪绿叶斑病毒Apple chlorotic leaf spot virus(ACLSV)、樱桃锉叶病毒Cherry rasp leaf virus(CRLV)、桃花叶病毒Peach mosaic virus(PMV)、李属坏死环斑病毒Prunus necrotic ringspot virus(PNRSV)、李痘病毒Plum pox virus(PPV)、李矮缩病毒Prunus dwarf virus(PDV)、樱桃绿环斑驳病毒Cherry green ring mottle virus(CGRMV)、杏假褪绿叶斑病毒Apricot pseudo-chlorotic leaf spot virus(APCLSV)、李树皮坏死茎纹孔伴随病毒Plum bark necrosis stem pitting-associated virus(PBNSPaV)和小樱桃病毒1号Little cherry virus 1(LchV1)的特异性引物进行RT-PCR检测。PCR结果显示仅HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV的扩增产物中得到了预期大小的目的片段,将目的片段克隆测序后,经NCBI BLAST比对发现,山东肥城桃分离物HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV与GenBank已报道分离物序列一致性均达90%以上。表明山东肥城桃已感染HSVd、PLMVd 2种类病毒和ACLSV、PNRSV、PBNSPaV 3种病毒。     

Apple necrotic mosaic virus,a novel ilarvirus from mosaic-diseased apple trees in Japan and China

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.     

Rose mosaic disease: symptoms induced in roses by graft inoculation with both prunus necrotic ringspot and apple mosaic viruses

Symptoms induced in rose by single isolates of the cherry serotype of prunus necrotic ringspot virus (PNRSV) and an apple serotype (apple mosaic virus; ApMV) were characteristically different, and appeared at different times throughout the growing season according to the ambient temperature. These features remained discrete, even in roses infected by both viruses and were shown by immunospecific electron microscopy to be a reliable indication of infection by either virus.
However, cross-protection between the two isolates was not reciprocal; mixed infections were established only when roses were simultaneously graft-inoculated with ApMV and PNRSV, or when PNRSV-infected roses were supei-infected with ApMV. The significance of these results in relation to the possible natural occurrence of mixed infections in rose or of isolates of intermediate serotype is discussed.     

The coat proteins and putative movement proteins of isolates of Prunus necrotic ringspot virus from different host species and geographic origins are extensively conserved

A complete sequence for the RNA 3 of Prunus necrotic ringspot virus (PNRSV) is described (Genbank Accession U57046). Primers from this sequence were used to amplify both the movement protein and coat protein genes of 3 other isolates of PNRSV originating from different host species and geographic locations. Comparisons of these sequences with those of other published sequences for PNRSV and the closely related apple mosaic virus (ApMV) showed that both the movement proteins and coat proteins of isolates of PNRSV are extensively conserved irrespective of either the original host or the geographic origin. The movement protein and coat protein of ApMV and PNRSV are sufficiently conserved to suggest that these two viruses may have evolved from a common ancestor. The amino acid sequence of the two coat proteins shows areas of similarity and difference that would explain the serological continuum reported to occur among isolates of these two viruses. Nevertheless, the movement protein and coat protein of the two viruses are sufficiently different so that ApMV and PNRSV should be considered to be distinct viruses.     

4种番茄病毒多重RT-PCR检测及应用

<正>番茄是全世界栽培最为普遍的果菜之一,2011年世界番茄栽培面积约805万亩,年产量约3 773万t,我国是世界番茄种植大国之一,2011年面积96667公顷,产量约679万t,随着番茄种植面积不断扩大,番茄病毒病的危害逐年加重~([1])。世界范围内番茄除了已有的TMV抗病资源与育成的抗