同步检测为害百合种球3种检疫性病毒的多重RT-PCR方法

 The multiplex RT-PCR approach was developed for simultaneous detections of Arabis mosaic virus(ArMV),Strawberry latent ringspot virus(SLRSV)and Lily symptomless virus(LSV)from the imported lily bulbs.The results indicated that good specificity and sensitivity for simultaneous detection were obtained.The ultimate of RNA detection with three viruses mixture was 305 pg.The ultimate of RNA detection with ArMV,SLRSV and LSV were 156.0,13.4 pg and 1.12 ng respectively.This approach has potential to be used in quarantine of imports and exports.     

江西甘蔗花叶病病原的分子鉴定

 Sugarcane mosaic disease, caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Maize dwarf mosaic virus (MDMV) or Johnsongrass mosaic virus (JGMV) in Potyvirus, is one of the most important viral diseases of sugarcane. In the study, four primer pairs specific to SCMV, SrMV, MDMV and JGMV, respectively, were designed and used to detect 29 sugarcane leaf mosaic samples collected from 9 locations in Jiangxi province. The representative RT-PCR products were sequenced. The results showed that 22 samples were infected by SCMV, three by SrMV, and four were mix-infected by SCMV and SrMV. MDMV or JGMV were not identified in all samples. The result indicates that SCMV is the major pathogen of sugarcane mosaic disease in Jiangxi province, and SrMV is also a pathogen for the disease.     

进境豇豆种子携带种传病毒的检测与鉴定

 Imported cowpea seeds were detected with growing test, ELISA assay and RT-PCR method. The ELISA results showed that cowpea seedlings with symptoms reacted positively with antibody against Southern bean mosaic virus (SBMV). The 979 bp of fragment could be amplified from two positive ELISA samples using primers specific for Southern cowpea mosaic virus (SCPMV), and the sequence determination results proved that the pathogen existing in imported cowpea seeds was SCPMV. The positive ELISA results with SBMV antibody could be further confirmed by RT-PCR amplification with specific primers designed to amplify the coat protein gene and 3' noncoding region of SCPMV and SBMV. The RT-PCR method presented here was suitable for molecular identification of SBMV and SCPMV in entry-exit plant quarantine laboratories.     

应用MNP-RT-PCR方法检测黄瓜绿斑驳花叶病毒

 A novel RT-PCR method integrated with Magnetic Nano Particles (MNP), MNP-RT-PCR, was set up for detection of Cucumber green mottle mosaic virus (CGMMV). After the virus particles in crude sap were concentrated by MNP, viral RNAs were released and were detected by RT-PCR. CGMMV could be detected in as less as 10 ng watermelon leaf materials. Compared with normal RT-PCR, the method decreased the inhibitors of plant material and steps for extracting RNA, and also increased the sensitivity of RT-PCR detection in less time. The method is simple and suitable for quick detection of plant virus in a large number of samples.     

藜草花叶病毒外壳蛋白基因的克隆与RT-PCR检测

 The nucleotide sequence of coat protein (cp)gene of Sowbane mosaic virus (SoMV)was determined. The cp gene of SoMV consists of 726 nucleotides and encodes a putative protein of 241 amino acid re-sidues. Sequence comparison showed that SoMV was most closely related to Rubus chlorotic mottle virus compared to other sobemoviruses. A primer pair was designed for the detection of SoMV based upon the determined cp nucleotide sequence. An expected fragment with 510 bp could be obtained from SoMV, while no specific band was observed from the healthy control. The result showed that the RT-PCR assay with the primer pair was suitable for the specific detection of SoMV.     

蚕豆染色病毒CPS基因的序列测定及RT-PCR检测方法的建立

 The nucleotide sequence of small coat protein (CPS) gene of Broad bean stain virus (BBSV) was determined and compared with other comoviruses. The CPS gene of BBSV consisted of 687 nucleotides and encodes a putative protein of 228 amino acid residues. The CPS sequence of BBSV and those of other comoviruses shared identities of 36.5%-58.9% and 35.2%-70.3% at the nucleotide and amino acid levels, respectively. The RT-PCR method specific for BBSV detection was developed based on the determined CPS sequence. The RT-PCR assay presented here allows, for the first time, rapid and specific detection of BBSV.     

广东番茄上检测到Tospovirus病毒

 Some tomato samples possibly infected by tospovirus in Guangdong were detected with indirect ELISA and RT-PCR. The results showed that the virus infected tomato did not react with the antiserum of Tomato spotted wilt virus (TSWV), but about 500 bp fragment of RT-PCR shared 83%-84% nucleotide identities with N gene of those reported tospoviruses. The phylogenetic tree of the N gene fragment compared with those of other tospoviruses indicated that the virus infected tomato was belonged to Tospovirus.     

稻曲病菌薄壁分生孢子制备的影响因素分析

本文研究了培养基、温度、振荡速度等因素对稻曲病菌Ustilaginoidea virens薄壁分生孢子产孢量的影响。结果表明,稻曲病菌薄壁分生孢子在培养第7天基本达到最大孢子量;该菌最适宜产孢的培养基为马铃薯煮汁,在煮汁中添加蔗糖可大幅提高产孢量;适宜的产孢温度为26~28℃;静止培养不利于产孢,振荡培养有利于产孢,并表现为转速越高产孢量越多;光照条件对产孢量没有影响。     

A more sensitive and rapid multiplex RT-PCR assay combining with magnetic nanobeads for simultaneous detection of viruses in sweet potato

An improved multiplex RT-PCR assay combined with magnetic nanobeads (MNB-RT-PCR) was developed for simultaneous detection of four sweet potato viruses, Sweet potato virus G (SPVG), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC) and Sweet potato chlorotic fleck virus (SPCFV). Four primer pairs specific for each virus were designed and the corresponding PCR products were 169, 357, 516 and 900 bp in length for SPVG, SPFMV, SPVC and SPCFV, respectively. The specificity of the method was tested using different combinations of virus templates, and the identities of the amplification products were confirmed by sequencing. The limits of detection for all four viruses by single and multiplex MNB-RT-PCR assays were comparable. The assay was further evaluated using laboratory and field samples compared with a conventional CTAB-RT-PCR assay, and the comparative results showed that the MNB-RT-PCR assay was more rapid and sensitive. These results suggest that the multiplex MNB-RT-PCR assay is an effective and preferable method for virus detection in sweet potato.     

利用小RNA测序技术鉴定吉林省甘薯病毒

在吉林省7个主要甘薯种植区共采集85份甘薯叶片样品,利用小RNA深度测序技术对混合样品进行检测,经RT-PCR和测序验证,鉴定出样品中存在10种病毒,包括6种RNA病毒和4种DNA病毒。分别是马铃薯Y病毒科马铃薯Y病毒属的甘薯羽状斑驳病毒Sweet potato feathery mottle virus (SPFMV)、甘薯潜隐病毒Sweet potato latent virus (SPLV)、甘薯G病毒Sweet potato virus G (SPVG)、甘薯C病毒Sweet potato virus C (SPVC)、甘薯2号病毒Sweet potato virus 2 (SPV2);长线形病毒科毛形病毒属的甘薯褪绿矮化病毒Sweet potato chlorotic stunt virus (SPCSV);双生病毒科菜豆金色花叶病毒属的甘薯曲叶病毒Sweet potato leaf curl virus(SPLCV);玉米线条病毒属的甘薯无症状1号病毒Sweet potato symptomless virus 1 (SPSMV1);花椰菜花叶病毒科杆状DNA病毒属的甘薯杆状DNA病毒B Sweet potato badnavirus B (SPBV-B)和甘薯隐症病毒Sweet potato pakakuy virus (SPPV)。     

中国甘薯病毒种类的血清学和分子检测

 2009～2010年,从我国18个省（市）采集了176份表现病毒病症状的甘薯样品。利用血清学、PCR和核苷酸序列测定的方法,对上述样品中的病毒种类进行了鉴定。血清学检测结果表明,供试样品中甘薯羽状斑驳病毒（SPFMV）的阳性率最高,达56.3%,其次为甘薯G病毒（SPVG）和甘薯类花椰菜花叶病毒(SPCaLV),阳性率分别为34.1%和33.5%。PCR和核苷酸序列测定结果表明,我国甘薯上至少存在SPFMV、SPVG、甘薯潜隐病毒（SPLV）、甘薯褪绿斑病毒（SPCFV）、甘薯褪绿矮化病毒（SPCSV）、黄瓜花叶病毒（CMV）、甘薯脉花叶病毒（SPVMV）和甘薯卷叶病毒（SPLCV）8种病毒。此外,供试样品中没有检测出甘薯轻斑驳病毒（SPMMV）,是否存在甘薯轻斑点病毒(SPMSV)、SPCaLV和C 6病毒尚不能确定。     

国家种质广州甘薯圃病毒种类鉴定及分析

为明确引起国家种质广州甘薯资源圃中病毒病的病毒种类及优势种,为甘薯种质安全保存提供支持,2017年从甘薯资源圃中未脱毒更新的盆栽苗和大田苗中采集155份具有不同病毒病症状的甘薯资源样品,利用PCR和RT-PCR检测技术对这些样品进行了17种病毒的分子检测.155份样品均有病毒检出,包括甘薯羽状斑驳病毒Sweet pot...     

3种甘薯病毒多重RT-PCR检测方法的建立及应用

正病毒病是引起甘薯品质降低和减产的重要原因之一,现已报道30多种能侵染甘薯的病毒~([1,2])。山东省是甘薯种植大省,病毒种类近10种~([3,4])。甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFM V)、甘薯潜隐病毒(Sweet potato latent virus,SPLV)是为害甘薯的主要病毒,在全国甘薯种植区广泛分布~([5,6])。甘薯病毒2(Sweet potato virus 2,SPV2)为Potyvirus的一个暂定种,多与同属的其他病毒混合侵染~([7])。多重PCR技术由Chamberian等~([8])1988年首次提出,可实现多基因的同时扩增,具有节省时间、提高效率的优点,已初     

我国发现由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒协生共侵染引起的甘薯病毒病害

甘薯病毒病害(Sweet potato virus disease,SPVD)是由毛形病毒属(Crinivirus)的甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)和马铃薯Y病毒属(Potyvirus)的甘薯羽状斑驳病毒(Sweet potato feathery mottle virus,SPFMV)协生共侵染甘薯引起的病毒病害[1].     

山东甘薯主要病毒的鉴定及多样性分析

为明确山东省甘薯病毒病发生现状,在重病区调查采样,通过鉴别寄主、电镜和分子检测技术明确主要病毒种类;并克隆病毒外壳蛋白基因序列,利用Mega 5.0构建系统进化树进行遗传分析。结果显示,巴西牵牛嫁接甘薯染病枝条后叶片黄化、褪绿及皱缩;病样组织中存在大量600~900 nm的线状病毒粒子和柱状内含体。24份病样中检测到甘薯羽状斑驳病毒、甘薯潜隐病毒、甘薯G病毒、甘薯曲叶病毒和甘薯褪绿矮化病毒5种病毒,其中23份为复合侵染,存在11种侵染类型。遗传分析显示山东省甘薯羽状花叶病毒主要为EA、O和C株系,甘薯潜隐病毒与周边省份分离物相近,甘薯G病毒与中国海南和美国分离物相近,甘薯曲叶病毒分属3个株系。表明山东地区甘薯病毒种类繁多,侵染模式复杂,病毒遗传结构具有多样性。     

Interactions between a crinivirus, an ipomovirus and a potyvirus in coinfected sweetpotato plants

Novel and severe symptoms of chlorosis, rugosity, leaf strapping and dark green islands, designated as sweetpotato severe mosaic disease (SPSMD), were caused by dual infection of Sweet potato mild mottle virus (SPMMV; Ipomovirus ) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus ) in three East African sweetpotato cultivars (Tanzania, Dimbuka and New Kawogo). The storage root yield was reduced by ∼80%, as compared with healthy plants under screenhouse conditions in Uganda. Plants infected with SPMMV or SPCSV alone showed nonsignificant or 50% yield reduction, respectively. SPCSV reduced resistance to SPMMV in sweetpotato, similar to the situation with resistance to Sweet potato feathery mottle virus (SPFMV; Potyvirus ) that breaks down following infection with SPCSV, followed by development of sweet potato virus disease (SPVD). In single virus infections with SPMMV and SPFMV or their coinfection, cvs Tanzania and Dimbuka were initially systemically infected, displayed symptoms and contained readily detectable virus titres, but new leaves were symptomless with very low virus titres, indicating recovery from disease. In contrast, cv. New Kawogo remained symptomless and contained low SPMMV and SPFMV titres following graft inoculation. These moderate and high levels of resistance to SPMMV and SPFMV, respectively, were lost and cultivars succumbed to a severe disease following coinfection with SPCSV. The synergistic interactions increased titres of SPMMV and SPFMV RNA by ∼1000-fold as quantified by real-time PCR, whereas SPCSV titres were reduced twofold, indicating an antagonistic interaction. Coinfection with SPMMV and SPFMV caused no detectable changes in virus titres or symptom severity.