纳米磁珠结合实时荧光RT-PCR技术检测大丽花潜隐类病毒

正大丽花潜隐类病毒(Dahlia latent viroid,DLVd)是Verhoeven et al.(2013)用Return-PAGE和s-PAGE技术从大丽花中鉴定出的一种新类病毒。根据2015年国际病毒分类委员会分类报告,DLVd为马铃薯纺锤形块茎类病毒科啤酒花矮化类病毒属成员。该病毒单独侵染时,大丽花没有明显的症状,其与马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTVd)复合侵染时可使大丽花叶片卷曲(Tsu-     

应用酶联免疫吸附试验法鉴定几种主要马铃薯病毒

制备了PVX,PVY_o,PVY~n,PVA,PVS,PVM,PLRV和TRV等8种主要马铃薯病毒抗体的辣根过氧化物酶结合物,在检测黑龙江省马铃薯主栽品种克新一、二、三、四号等品种的植株样品1155份和800余份正在脱毒中的试管苗样品中,发现上述8种马铃薯病毒在几个主栽品种中的侵染率较高,而且大多数感病植株是由3至7种病毒复合侵染,并首次查明本省存在PVY~n和PVM病毒。对不带上述8种病毒的样品又用双向反向聚丙烯酰胺凝胶电泳鉴定带马铃薯纺锤块茎类病毒(Potato spindle Tuber Viroid)状况,现已获得无上述8种病毒和类病毒的种植材料。     

基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒

为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。     

上海崇明水仙病毒病病原种类鉴定

 2005~2007年对上海崇明水仙病毒病的种类进行了调查和鉴定。崇明水仙病毒病田间主要表现为花叶、斑驳、黄条和白条等多种症状,以花叶和斑驳为主。采用ELISA和RT-PCR方法,从36个水仙样本上检测到水仙普通潜隐病毒(NCLV)(GenBank登录号:EU200454)、水仙退化病毒(NDV)(GenBank登录号:EU200456);此外,有两类分离物被初步鉴定为新病毒,暂命名为水仙斑驳相关病毒(NMaV)(GenBank登录号:EU182651)和水仙白条相关病毒(NWSaV)(GenBank登录号:EU182652)。4类病毒的总检出率分别为11.1%、13.9%、100%和2.8%,其中NMaV为危害水仙的优势病毒。约20%的样本检测出有2种以上病毒的复合侵染,但是未检测出Potexvirus属的病毒。     

RT-PCR技术对宁夏马铃薯脱毒种薯病毒检测的研究

病毒病是影响马铃薯生产的重要病害,为了解宁夏地区马铃薯种薯生产中的病毒发生情况,在银川市、固原市和西吉县不同种植模式的脱毒种薯生产基地,对不同品种的原原种、原种、一级种在不同生育期随机采集样品,利用RT-PCR检测技术对4种马铃薯主要病毒PVX、PVY、PVS、PLRV和类病毒PSTVd进行检测。结果表明:PVX、PVY、PVS、PLRV在3个基地均有不同程度检出,成熟期病毒检出率分别为35%、28%、35%和34%,未检出PSTVd;4种病毒检出率在马铃薯现蕾期以后呈现显著性增长;病毒检出率随着种薯繁育级别增加差异显著;6个马铃薯品种均为感病品种;大田种植种薯的病毒检出率显著高于防虫网室。     

梨泡状溃疡类病毒的RT-PCR检测及序列分析

正梨泡状溃疡类病毒(Pear blister canker viroid,PBCVd)为马铃薯纺锤块茎类病毒科(Pospiviroidae)苹果锈果类病毒属(Apscaviroid)成员~([1])。该类病毒在自然条件下主要侵染野生和栽培的梨和苹果~([2~4]),梨和榅桲多数品种感染PBCVd后无明显症状,通过嫁接传染该类病毒可诱导梨指示植物     

马铃薯种苗复合感染病毒多重RT-PCR同步快速检测

 基于马铃薯病毒ssRNA的快速制备方法,根据马铃薯病毒CP基因序列设计PVX、PVS、PVY和PLRV特异性引物对、P1基因序列设计PVA特异性引物对,建立了多重RT PCR快速检测体系,可以同步检出复合侵染马铃薯种苗主要病毒,灵敏度比传统的ELISA至少高100倍。结合生物活性稳定剂研制的固相化检测试剂盒,已用于四川和重庆等15个县市田间与苗圃248个马铃薯显症或无症样品的实际检测,表明四川和重庆地区2~3种马铃薯病毒往往复合侵染(PVX、PVA和PVS三种病毒复合侵染或PLRV和PVY二种病毒复合侵染)。     

甘肃省马铃薯主要病毒病发生情况调查

2015年-2016年,在甘肃省10个地市24个马铃薯主栽县(区)146个生态区域(乡镇)采集了757份具有典型症状的马铃薯样品,应用DAS-ELISA法进行检测,筛查6种主要病毒(PVX、PVY、PLRV、PVA、PVS和PVM)。结果表明:631份样品检测到病毒,PVS的检出率最高,达47.03%,PVY次之,为33.82%,PVA最低,只有0.63%;发生复合侵染的病毒主要为PVY+PVS,复合侵染率达到10.13%,三种病毒复合侵染主要是PVY+PVS+PVM;病毒种类和感病程度与品种、地域有关。     

多重RT-PCR同时检测6种马铃薯病毒及1种类病毒

病毒病是限制我国马铃薯生产的重要因子之一。本研究通过引物设计和体系优化建立了一套多重RT-PCR检测方法，可以在一个反应体系中同时检测6种马铃薯病毒和1种马铃薯类病毒以及1个马铃薯内参基因，包括马铃薯Y病毒(potato virus Y, PVY)、马铃薯M病毒(potato virus M, PVM)、马铃薯S病毒(potato virus S, PVS)、马铃薯X病毒(potato virus X, PVX)、马铃薯A病毒(potato virus A, PVA)、马铃薯卷叶病毒(potato leaf curl virus, PLRV)、马铃薯纺锤块茎类病毒(potato spindle tuber viroid, PSTVd)和细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,CoxⅠ)基因的特异片段，产物大小依次为181、226、275、565、630、681、359、500 bp,符合理论预期。采用建立的方法以相应病毒和类病毒的质粒作模板进行检测，其检测灵敏度在1.6×10^-3～1.8×10^-1 ...     

“国际类病毒及卫星病毒学术研讨会”概况及主要研究进展

本文旨在对举办国际类病毒及卫星病毒学术研讨会的组织筹备、工作经验等进行交流,对大会学术交流所涉及的新类病毒/环状RNA分子的鉴定及其风险评估、类病毒及卫星RNA侵染导致寄主的反应、类病毒的遗传多样性和复制以及类病毒的检测技术及防治方法等四方面内容的研究亮点及新进展等进行总结,希望对广大科研工作者有一定的帮助。     

Potato germplasm poses the highest risk of introducing potato spindle tuber viroid in potatoes in the Netherlands: analysis and evaluation of an outbreak in potato breeding

In 2014, potato spindle tuber viroid (PSTVd) was identified in a potato clone originating from a breeding company in the Netherlands. This clone was submitted for micro propagation and therefore tested for PSTVd and a number of other pathogens. This finding of PSTVd initiated actions to track and eradicate the infections. In addition to the finding at the breeding company, PSTVd was also found at a research institute. At both locations the viroid was eradicated following extended testing and discarding of infected plants. Additional surveys including testing of each individual plant in all crossing glasshouses and random samples of pre-basic and basic seed potatoes, revealed no further infections in the Netherlands. This result concurred with the fact that mechanical spread of PSTVd in the field is not likely under climatic conditions in the Netherlands. Therefore, vegetative propagation seems the most important pathway for maintaining and spreading of PSTVd. Based on the evaluation of this outbreak, it was concluded that potato germplasm poses the highest risk of introducing this viroid in potatoes in the Netherlands.     

不同探针大小、标记物及反应底物对马铃薯类病毒杂交检测的影响

 灵敏可靠地检测马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTVd)对脱毒种薯的生产具有重要意义。本研究探索了影响核酸杂交检测技术的关键因素。通过基因克隆技术构建了插有PSTVd全长单体、双体及片段的载体。分别以地高辛和同位素为标记物,利用PCR和转录标记技术制备cDNA和RNA探针。比较探针大小、标记物、标记方法、反应底物等对检测灵敏度的影响。结果显示,以地高辛为标记物,利用PCR标记制备的PSTVd双体cDNA探针,在以CDP-Star为底物,通过在柯达X-OMAT BT胶片进行化学发光反应来分析结果的检测灵敏度最高,可以检测到0.05 pg总RNA中的PSTVd,是国外报道检测灵敏度的500倍。利用核酸斑点杂交技术检测PSTVd具有灵敏度高,一次可检测样品数量多等特点,对于大规模PSTVd检测更加方便可行。     

An outbreak of Potato spindle tuber viroid in tomato is linked to imported seed

In 2011, an outbreak of the quarantine-regulated pathogen Potato spindle tuber viroid (PSTVd) occurred in a commercial glasshouse-grown tomato crop in Queensland, Australia. Phylogenetic studies showed that the genotype of this isolate grouped in a cluster of PSTVd genotypes from tomato and Physalis peruviana, and exhibited an interesting mutation (U₂₅₇→A) that has previously been linked to lethal symptom expression in tomato. Transmission studies showed that the viroid could be mechanically transmitted from crushed fruit sap, but not from undamaged fruits. A low rate of asymptomatic infection was determined for plants in the affected glasshouse, demonstrating the efficacy of using symptoms to detect PSTVd infections in tomato. No PSTVd infections were detected in solanaceous weeds located outside of the infected glasshouse, excluding them from playing a role in the viroid epidemiology. Monitoring and subsequent testing of new tomato crops grown in the facility demonstrated successful eradication of the pathogen. A trace-back analysis linked the outbreak of PSTVd to an infected imported tomato seed-lot, indicating that PSTVd is transmitted internationally through contaminated seed.     

Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles

Potato spindle tuber viroid (PSTVd) was transmitted by Myzus persicae to Physalis floridana from P. floridana plants that also were infected with potato leafroll luteovirus (PLRV), whereas it was not transmitted by aphids from plants infected with PSTVd alone. Dot-blot hybridisation was used to detect PSTVd. The results indicate that PLRV can assist PSTVd in its transmission by M. persicae. Doubly infected, aphid-inoculated P. floridana plants from the previous experiment were used as the source plants in aphid transmission tests to the tomato cv. Rutgers, P. floridana and Datura stramonium. PSTVd was detected in 17 of 30 plants of tomato. The viroid was not detected by dot-blotting in any plant of P. floridana and D. stramonium in this experiment, but it was recovered from some plants by sap inoculation of the Rutgers plants. Treatment with RNase A of PLRV preparations purified from doubly infected plants indicated that PSTVd was encapsidated by PLRV particles.     

Epidemiological evidence that vegetatively propagated, solanaceous plant species act as sources of Potato spindle tuber viroid inoculum for tomato

In autumn 2006 in the Netherlands, Potato spindle tuber viroid (PSTVd) infections were detected in 42·3 and 71·9% of professionally grown lots of Brugmansia spp. and Solanum jasminoides respectively. The infected lots contained 73 985 and 431 374 plants, respectively, demonstrating the presence of many potential viroid sources for tomato ( Solanum lycopersicum ). PSTVd was identified in cultivars of Brugmansia × candida , B. × flava , B. sanguinea , B. suaveolens and unspecified Brugmansia species/cultivars. Most infected lots of Brugmansia spp. originated from a single Dutch nursery; most infected lots of S. jasminoides originated abroad. Sequence analysis revealed that the PSTVd genomes from Brugmansia spp. contained an average of 360 nt, whereas all genomes from S. jasminoides except one consisted of 357 nt. Furthermore, the collective PSTVd genotypes showed polymorphism at four or more positions, except for two cases in which genotypes from Brugmansia spp. and S. jasminoides were identical. Phylogenetic studies showed that PSTVd genotypes from Brugmansia spp. and S. jasminoides grouped apart from each other and from PSTVd isolates from potato ( Solanum tuberosum ) and Physalis peruviana . The PSTVd genotypes from tomato did not form a separate cluster, but were dispersed over clusters of vegetatively or partly vegetatively propagated plant species, i.e. potato, P. peruviana and S. jasminoides . Moreover, mechanical inoculation of the predominant PSTVd genotypes from S. jasminoides to tomato was successful. These results provide evidence that vegetatively propagated, solanaceous plant species have been sources of infection for tomato crops in the past.     

Comparison of direct-RT-PCR and dot-blot hybridization for the detection of Potato spindle tuber viroid in natural host plant species

Potato spindle tuber viroid (PSTVd) is an EPPO A2-listed quarantine pathogen and its detection in large scale surveys requires complex decision schemes. In this study, a simple and rapid application of direct-RT-PCR was evaluated together with dot blot hybridization for the detection of PSTVd in dormant potato tubers harvested from primary infected plants, as well as in tomato and solanaceous ornamental plants. In all infected dormant potato tubers tested, both direct-RT-PCR and dot blot hybridization detected two different PSTVd isolates, with direct-RT-PCR being ten times more sensitive than dot blot. Similarly, in infected tomato and Brugmansia spp., PSTVd was detected by direct-RT-PCR with higher sensitivity compared to that of dot blot hybridization. However, in Brugmansia spp., a ten-fold decrease of the typical working concentration of the sap was required for an unequivocal detection of the viroid by direct-RT-PCR. The potential to use direct-RT-PCR for routine PSTVd examination is discussed.     

Development of an EU protocol for the detection and diagnosis of Potato spindle tuber pospiviroid*

The work described here formed part of the EU SMT DIAGPRO project, to develop diagnostic protocols for 18 regulated pests. The Potato spindle tuber pospiviroid (PSTVd) protocol was developed primarily for testing in vitro‐ and glasshouse‐grown potato plants for the purposes of post‐entry quarantine and the production of pathogen‐tested nuclear stock. After a performance audit of methods used by 12 laboratories in Europe and America by ring testing, four methods were chosen for multilaboratory validation. For most laboratories, the detection limits were 10–20 mg of PSTVd‐infective tissue for R‐PAGE; 0.25–0.5 mg for DIG‐probe; 0.062 mg for RT‐PCR; and 0.0155 mg for TaqMan (this was the lowest weight of infective tissue tested). Some laboratories were able to extend the detection limit to 0.0155 mg for DIG‐probe and RT‐PCR. The DIG‐probe and R‐PAGE are recommended as primary detection methods, with confirmation of viroid presence by any of the four validated detection methods. Specific diagnosis requires the viroid to be sequenced. Other methods may be used for primary detection, providing that they preferably detect all PSTVd isolates and other Pospiviroids that have the potential to infect potato, and detect viroid in at least 1/10 of the tissue weight normally tested per plant.     

The distribution of potato spindle tuber viroid in potato plants and tubers1

Potato spindle tuber viroid (PSTV) in potato plants was investigated by ‘return’ gel electrophoresis. The experiments were carried out under quarantine conditions in the greenhouse with primarily and secondarily infected plants. The PSTV content in different plant parts was estimated by the intensity of the viroid band in polyacrylamide gel. The results showed a decrease of viroid content from the upper to the lower parts of the plant. In both primarily and secondarily infected plants, PSTV was reliably detected in the top leaves, but less so in the lower leaves. In four out of ten secondarily infected plants, PSTV was found in the roots. In dormant tubers, the bands were more intense with samples obtained from the rose end and the heel than from those obtained from the medullary tissue. With one exception, all 64 tubers from 26 primarily infected plants were infected with PSTV.