从海南省杂草胜红蓟和假马鞭上检测到粉虱传双生病毒

 利用三抗体夹心ELISA(TAS-ELISA)及PCR检测的方法对采自海南的胜红蓟(Ageratum conyzoides)和假马鞭(Stachytarphetajamaicensis)的5个病样进行了检测,表明均为粉虱传双生病毒。PCR扩增产物克隆后进行序列测定,结果表明存在2类双生病毒,其中样品Hn2存在2类病毒的复合侵染。这是在海南首次报道存在有粉虱传双生病毒。     

中国番木瓜曲叶病毒南宁分离物的基因组结构特征

 从广西南宁田间表现曲叶症状的番木瓜植株上分离到病毒分离物G4,经三抗体夹心ELISA (TAS-ELISA)检测,G4与粉虱传双生病毒的抗体呈阳性反应。对G4 DNA-A全序列测定和分析表明,G4 DNA-A全长2 748个核苷酸,共编码6个ORFs。同源性比较及系统进化关系分析表明,G4 DNA-A与在亚洲发现的粉虱传双生病毒关系较近,其中与我国报道的中国番木瓜曲叶病毒(PaLCuCNV)同源性最高,达到98.0%。进一步比较发现,G4 DNA-A编码的AV1、AV2、AC1、AC2、AC3和AC4与PaLCuCNV相应ORFs的氨基酸同源性分别为98.4%、95.7%、97.5%、97.8%、94.1%和94.6%,表明G4应属于PaLCuCNV的一个分离物。G4编码的ORFs与中国胜红蓟黄脉病毒(AYVCNV)、辣椒曲叶病毒(PepLCV)及烟草曲茎病毒(TbCSV)有较高的氨基酸同源性,可能起源于共同的祖先。利用DNA-B及卫星DNAβ的保守引物均未能从G4分离物中扩增出相应的组分。     

烟粉虱中甘薯双生病毒(sweepoviruses)半巢式PCR快速检测技术的建立与应用

甘薯双生病毒(sweepoviruses)是侵染甘薯的一类重要病毒,通过烟粉虱以持久方式传播,我国甘薯上至少存在8种甘薯双生病毒.本研究根据我国已报道的8种甘薯双生病毒基因组保守区设计了一组引物,建立了单头烟粉虱中甘薯双生病毒的半巢式PCR快速检测方法.特异性和灵敏性分析结果表明,半巢式PCR具有较高的特异性和灵敏性,...     

单抗I-ELISA和TAS-ELISA检测百合无症病毒的研究

 以抗百合无症病毒(Lily symptom less virus,LSV)的单克隆抗体为核心,建立了间接ELISA (I-ELISA)和三抗体夹心ELISA (TAS-ELISA)的检测方法。I-ELISA检测体系中,病叶汁液和单克隆抗体腹水的工作浓度分别为1:20和1:6 000,对病叶汁液的检测灵敏度达到了1:2 560,可检测到提纯病毒绝对量为1.35 ng。TAS-ELISA检测体系中捕获抗体和单克隆抗体腹水的工作浓度分别为1:200和1:6 000,检测病叶汁液的灵敏度达到了1:5 120,对于提纯病毒可检测到0.68 ng。使用美国Agdia公司双抗体夹心ELISA (DAS-ELISA)检测试剂盒对病叶汁液的检测灵敏度为1:2 560,提纯病毒检出量1.35 ng。用3种ELISA方法检测了采自浙江省丽水市的46个田间样品,I-ELISA、TAS-ELISA和DAS-ELISA测出的阳性样品数分别为19、21和18个,阳性率为41%、46%和39%。灵敏度检测和田间样品检测结果显示,TAS-ELISA的灵敏度高于DAS-ELISA和I-ELISA。相同样品I-ELISA所测出的OD₄₀₅值和P/N值普遍高于DAS-ELISA,表明LSV单抗比多抗具有更强的特异性和更高的灵敏度。     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

灰飞虱体内水稻条纹病毒的检测

为评价灰飞虱体内水稻条纹病毒检测方法的适用范围,利用已经制备的水稻条纹病毒(Rice stripe virus,RSV)的多克隆抗体SV21和单克隆抗体3B9,采用多孔板间接ELISA、DIBA和Western blotting等三种方法进行单头灰飞虱Laodelphax striatellus Fallén(SBPH)体内RSV检测。结果表明,检测灵敏度以多孔板间接ELISA最高,其次为DIBA,Western blotting最低;用单克隆抗体3B9检测灵敏度高于多克隆抗体SV21。RT-PCR、IC-RT-PCR和DB-RT-PCR三种方法中,RT-PCR对单头灰飞虱稀释到400倍可检测到病毒;IC-RT-PCR在多克隆抗体SV21稀释浓度大于500倍时检测不出RSV,单克隆抗体3B9稀释浓度大于800倍时检测不到RSV;DB-RT-PCR检测结果显示,单头灰飞虱在稀释400倍后均无阳性反应。     

用滚环扩增技术检测双生病毒

 本文报道了利用滚环扩增技术(rolling circle amplification,RCA)放大双生病毒的靶核酸信号,进而利用限制性内切酶片段长度多态性(RFLP)检测不同类型双生病毒的新技术体系。研究结果表明,利用RCA-RFLP对于已知的单组份和双组份以及带有卫星的菜豆金色花叶病毒属病毒都能够有效检测,对采自浙江杭州地区的田间样品进行RCA-RFLP检测的结果与利用简并引物PA和PB进行PCR扩增的结果相吻合。相比传统的PCR检测方法,用RCA-RFLP体系检测双生病毒灵敏性更高,操作更简便。     

广西粉虱传染类植物双生病毒调查

广西粉虱传染类植物双生病毒调查植物双生病毒（Ｇｅｍｉｎｉｖｉｒｕｓ）具有独特的球状对生病毒形态和单链ＤＮＡ，由于其经济和理论研究上的重要性，近年成为病毒学研究的热点之一，但是，国内有关此组病毒的报道极少。笔者自１９９１年以来在广西对粉虱传染类植物双生...     

侵染新疆加工番茄的中国番茄黄化曲叶病毒 DNA-A的基因组特征

 从新疆加工番茄上分离到病毒分离物XJ26-4,对其基因组DNA-A 全序列测定表明,XJ26-4 DNA-A 全长2 737 个核苷酸(GenBank 登录号:FN985163),具有典型的双生病毒基因组特征。进一步序列比较发现,XJ26-4 DNA-A 与中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus, TYLCCNV)各分离物的同源性最高,达到91. 2% ~ 99. 5% ,而与其他双生病毒的序列相似性均在79. 5% 以下,表明XJ26-4 是TYLCCNV 的一个分离物。这是首次明确新疆加工番茄受到粉虱传双生病毒的侵染。     

Virus transmission studies and diagnostic detection of four begomovirus isolates in selected crop hosts and in Bemisia tabaci biotype B*

Virus transmission studies were conducted under glasshouse conditions using the vector Bemisia tabaci biotype B to determine how effectively isolates of the begomoviruses Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl Bangalore virus (ToLCBV) could be transmitted to phaseolus bean, capsicum and tomato test plants, the latter host used as a positive control for transmission. Diagnostic detection of viruses in these host crops and vector was also evaluated. Polymerase chain reaction (PCR) detection of TYLCV in bean cv. Wade and capsicum cv. Bellboy was achieved 4 weeks after fumigation in asymptomatic plants. Detection of TYLCV in tomato controls was achieved 2 weeks after fumigation with improved frequency of detection at 4 weeks. PCR was found to be a more sensitive method than triple‐antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA) for the detection of TYLCV isolates in all hosts. ToLCBV was detected by PCR and TAS‐ELISA in bean. TYLCV was also detected by PCR in the vector, with a novel internal positive control. This work was carried out to facilitate the development of a diagnostic protocol for the begomoviruses causing tomato yellow leaf curl under the EU SMT programme project –‘Diagnostic protocols for organisms harmful to plants’ (DIAGPRO).     

应用实时荧光PCR检测苹果果实球壳孢腐烂病菌

根据苹果果实球壳孢腐烂病菌(Sphaeropsis pyriputrescens Xiao&J.D.Rogers)ITS区序列设计特异性引物和TaqMan荧光探针,建立了苹果果实球壳孢腐烂病菌的实时荧光PCR检测方法。该方法能够特异性检测苹果果实球壳孢腐烂病菌,供试的目标菌株检测结果为阳性,而苹果轮纹病菌、苹果炭疽病菌、苹果干腐病菌和苹果褐腐病菌、苹果腐烂病菌等5种对照菌株检测结果为阴性。该方法具有快速、简便、准确的优点,适合于该病害的快速诊断和口岸检验检疫运用。     

梨火疫细菌实时荧光PCR和诱捕PCR-ELISA检测方法的建立

根据梨火疫细菌中独特而保守存在的质粒pEA29，设计了1对引物和3条探针，建立了实时荧光PCR检测方法和诱捕PCR-ELISA检测方法。实时荧光PCR采用带荧光标记的核酸杂交探针，边扩增边检测，步骤简单，不需PCR后处理，可避免假阳性和交叉污染；诱捕PCR-ELISA检测方法只需简单处理的样品就能检测，减少了核酸不纯出现的漏检，由于增加了核酸杂交探针，可不需凝胶电泳EB染色检测，不会出现假阳性问题。     

Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA, symptomatology, biological indexing and cross-protection tests

Serological detection of Citrus psorosis virus (CPsV) by direct tissue blot immunoassay (DTBIA) and by double (DAS) and triple (TAS) antibody sandwich ELISA, was compared in samples from various citrus varieties growing in the glasshouse and in the field. In young shoots and leaves, CPsV was readily detected by the three procedures, whereas DTBIA detection in old leaves was less consistent. DTBIA detection and ELISA readings in nine different citrus varieties were similar, suggesting that CPsV accumulates to equivalent levels in all of them. In infected field trees from Spain or Italy, CPsV was consistently detected by TAS ELISA, even in samples of old leaves in winter, whereas DTBIA detection in the same trees was reliable only when using young shoots. Detection of CPsV by DTBIA and by DAS and TAS ELISA in previously untested field trees correlated perfectly with psorosis diagnostics based on biological indexing, specifically with the capacity of those sources to cross-protect against challenge inoculation with psorosis B. Some trees without bark scaling were shown to be psorosis-infected by biological indexing and to contain CPsV by serological tests; other trees showing psorosis-like bark or leaf symptoms in the field were shown to be psorosis-free by biological indexing and also CPsV-free by serology. This is the first time that the presence of CPsV has been correlated with psorosis infection as diagnosed by biological indexing.     

苜蓿花叶病毒ELISA检测方法的建立及其在大豆病毒调查和抗性资源鉴定中的应用

苜蓿花叶病毒(alfalfa mosaic virus, AMV)是一种世界性分布、宿主范围广、具有严重危害性的植物病毒,能引起大豆的严重病害。本研究利用原核表达的AMV CP蛋白制备的抗血清,建立了高效、准确的AMV间接ELISA检测方法,并应用于病害调查和抗性鉴定,结果表明制备的3份抗血清对重组蛋白和AMV感染的大豆植物粗提液的效价均达到256 000倍,血清特异性分析结果显示3份抗血清仅识别感染AMV的大豆叶片,不识别感染大豆花叶病毒(soybean mosaic virus, SMV)的大豆叶片。通过建立的AMV间接ELISA与常规RT-PCR同时对采集的50份疑似感染AMV的大豆样品进行检测,有46份样品检测结果一致,符合率达92%。利用建立的AMV ELISA方法和课题组已建立的SMV ELISA方法对吉林省大豆主产区的大豆样品进行病毒检测的结果表明,病毒检出率为38.30%,SMV的检出率达30.85%,AMV的检出率达17.06%,复合侵染率为9.61%。对接种AMV的40个大豆品种进行抗性鉴定,结果显示40份大豆全部感染AMV,但是病毒载量存在差异,部分品种表现出AM...     

植原体TaqMan探针实时荧光PCR检测鉴定方法的建立

 本研究成功建立了植原体分类鉴定和检测的TaqMan探针实时荧光PCR方法,该方法根据植原体16S rDNA保守区设计了1个TaqMan广谱探针和3个植原体组间点突变特异性探针,并对9种植原体和5种细菌以及3个植物样本进行实时荧光PCR。结果表明,用广谱探针可检测到所有植原体产生荧光信号,而细菌不产生荧光信号。当用植原体组间特异性探针检测时,仅能检测到该组植原体产生荧光信号,检测的敏感性比常规的PCR-电泳检测高约100倍、检测速度有较大提高。由于PCR产物是荧光探针检测,本方法特异性强,并可以用组特异探针直接确定植原体种类。实验采用完全闭管检测,降低了污染机会。本研究为其它原核生物、特别是不能培养菌、难培养菌的检测鉴定和分类提供了新方法。     

南芥菜花叶病毒的几种PCR检测方法的建立和比较研究

 以进境种球中截获的带毒洋水仙和郁金香为试验材料,建立了ArMV的免疫捕获RT-PCR、巢式PCR和Real-time PCR方法,并比较了几种检测方法的灵敏度。DAS-ELISA的检测灵敏度较低,为1mg洋水仙或10mg郁金香带毒种球,而各种PCR方法的灵敏度可高于DAS-ELISA 100倍以上,其中Real-time PCR检测的灵敏度最高,可从20ng洋水仙或2μg郁金香的带毒种球中检出ArMV。鉴于DAS-ELISA灵敏度较低,建议在用ELISA初筛时,如样品OD₄₀₅值与阴性对照OD₄₀₅值之比在2.0左右时需要再用分子方法加以确证,以防漏检。     

Occurrence of Citrus psorosis virus in Campania, southern Italy

Citrus psorosis virus (CPsV), genus Ophiovirus, is associated with a severe disease of citrus worldwide. Double antibody sandwich (DAS) ELISA using a polyclonal antiserum, and triple antibody sandwich (TAS) ELISAs, employing the IgG monoclonal antibody (mab) 13C5, and the IgM mab 2A3, were used to detect CPsV in orchards of different citrus varieties in Campania, southern Italy. TAS ELISA with 13C5 detected all the infections detected by DAS ELISA. Overall, 14% of trees younger than 15 years were positive, but only 1% of older trees, suggesting that infected propagating material has been increasingly used in recent years, in the absence of certification. Highest infection rates were in younger trees of sweet orange (22.8%) and clementine (18.6%). CPsV could easily be detected at all seasons of the year tested (June–January); these and earlier results indicate that TAS ELISA using 13C5 is a sensitive, broad-spectrum and reliable diagnostic method useful for routine tests and certification programmes. Of 44 field isolates responding strongly to DAS ELISA and 13C5-TAS ELISA, mab 2A3 gave similar results with 29 isolates, but gave low values with the others, thus providing a degree of differentiation among isolates. To confirm that the ELISA tests were indeed detecting CPsV, samples of 42 ELISA-positive plants were analysed by ISEM in a blind test, and in 38 of these, characteristic virus particles were clearly seen. Although CPsV was frequently and consistently detected in the area sampled, bark scaling symptoms were not seen: possible reasons for this are discussed.     

Production of monoclonal antibodies to potato virus YNTN strain and their use for strain differentiation

Four mouse monoclonal antibodies (MAbs) against potato virus Y (PVY) were produced. MAb 4C1 reacted with four isolates of PVY^NTN and only very weakly with one isolate of the necrotic strain of PVY (PVY^N). It did not react with other isolates of the ordinary strain of PVY tested. MAb 2C9 reacted with all isolates tested and can be used to produce a specific diagnostic kit for routine PVY detection. Other MAbs had different specificities and reacted with isolates of various strains of PVY. MAbs did not react with seven other members of the Potyvirus group including potato virus A. A MAb-based ELISA, using MAb 4C1, was devised and shown to detect PVY^NTN specifically.