中山局从进镜花卉种球中检出3种病毒

2006年10～12月期间，中山检疫局技术中心植检实验室对从荷兰进境的2000多头百合种球和400多头剑兰种球进行检疫，利用RT—PCR分子检测方法，从样品中检出带有百合无症病毒和百合斑驳病毒（Lily mottle virus）复合感染的百合种球1株；在剑兰种球中检出2株带有菜豆黄色花叶病毒（Bean yellow mosaic virus），这是中山局首次检出这3种植物病毒，另外菜豆黄色花叶病毒也是广东辖区内首次检出的病毒。     

从盐田局送检的百合种球中检出百合斑驳病毒

2004年4月27日，深圳检疫局盐田分局从荷兰进口的百合种球中抽取样品送动植中心进行病毒检测。经隔离种植后，发现有一株百合叶片上出现斑驳、条纹、扭曲等症状，疑是百合斑驳病毒侵染。随后，采用百合无症病毒RT-PCR检测方法，特异性扩增得到一条大约500bp左右的特异性条带，这与期望结果一致。为了保证检测结果准确无误，又将RT-PCR扩增产物进行测序，测序结果与GenBank上已登录的百合斑驳病毒多聚蛋白基因保守区序列一致，也与预先设计的基因片断完全一致。从而表明，从荷兰进口的百合种球中确实携带有百合斑驳病毒（Uly mottle virus）。     

进境百合种球病毒不同检测方法初析

我国大量进口的百合种球易携带多种检疫性病毒及限定性有害生物,进境口岸实验室对百合种球病毒的检测尤为重要.以百合无症病毒(Lily symptomless virus,LSV)为对象,分别采用双抗体夹心酶联免疫吸附(DAS-ELISA)及反转录PCR(RT-PCR)方法进行检测,根据过程与结果,初步分析不同检测方法的特点...     

10种植物病毒的基因芯片检测技术研究

 本研究设计了10种植物病毒和各种对照的探针, 将探针固定在醛基化玻璃片基上制备基因芯片。用常规的Trizol法提取植物总RNA, 根据病毒的外壳蛋白基因或复制酶基因设计特异性引物, 经RT-PCR扩增相应区段并用Cy3-dCTP进行标记。将标记的PCR产物与芯片杂交, 扫描仪对杂交结果扫描, GenePix Pro 4.0软件对杂交图像进行分析。结果表明, 该芯片可以从病毒感染样本中检测到特异性识别信号, 检测灵敏度比RT-PCR高10~100倍, 所以, 基因芯片能对植物病毒作出快速、准确的检测。     

百合脱毒组培技术研究

百合为百合科百合属(Lilium)多年生球根植物,是国际上主要鲜切花之一。百合在生产中一般采用无性繁殖,而百合病毒可通过种球传播。因此,病毒一旦感染百合,即在种球内不断积累,最终导致种质严重退化,给百合种植业造成巨大的经济损失。迄今已发现引起百合种球退化的病毒及其类似病原多达12种,其中危害最严重的有百合潜隐病毒(Lily symptomless virus,LSV),郁金香碎色病毒(Tulip breaking virus,TBV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)。随着组织培养技术的日臻成熟,通过组培脱毒为解决百合种球病毒性退化问题提供有效途径。国内外对百合组培脱毒进行的系列研究表明,获得脱毒苗的方     

侵染厦门百合的百合无症病毒的鉴定及其分子检测

百合无症病毒(Lily symptomless carlavirus, LSV)属于香石竹潜隐病毒组(Carlavirus),是单链正义RNA病毒。基因组全长为8 394 bp,共有6 个开放阅读框(Open reading fragment,ORF),分别编码RNA依赖的RNA复制酶,三基因连锁结构, 外壳蛋白和16 kD核酸结合蛋白。主要侵染百合属、郁金香属植物,通常在百合上不产生明显症状,与黄瓜花叶病毒(Cucumber mosaic virus, CMV)复合侵染时,导致叶片出现坏死斑。LSV 是危害百合的重要病毒,在世界各地都有分布。近年来国内陆续有该病毒病的报道,其发病率一般在40％-50％,二代种球的带毒率在90％以上, 严重制约了我国百合鲜切花的产量和质量,目前LSV已成为各口岸进出口百合种球的主要检疫对象。     

花卉根部及基质中虫害的药剂处理试验

本文记录了一品红种苗根部、百合种球及其基质中虫害的主要种类,并分别用6种和4种农药对一品红及百合根部进行对比试验.结果表明辛硫磷对一品红种苗中的双翅目害虫效果较好,卵螨速杀和哒螨灵对百合种球中的刺足根螨效果较好.     

荷兰进口百合中检出百合无症病毒

本文采用DAS-ELISA、RT-PCR方法从隔离试种的进口荷兰百合中检出百合无症病毒,并通过对RT-PCR产物进行序列分析验证,确认进口的百合携带有百合无症病毒(LSV).     

香石竹环斑病毒的分子检测

试验具体研究了两类不同性质的ｃＤＮＡ探针对香石竹环斑病毒的检测效果。毒源由中国农业大学植物病毒室提供，繁殖于苋色藜Ｃｈｅｎｏｐｏｄｉｕｍａｍａｒａｎｔｉｃｏｌｏｒ上，提纯该病毒，然后抽提其核酸ＲＮＡ，将ＲＮＡ多聚腺苷化后，以Ｏｌｉｇｏ（ｄＴ）为引物，反转录合成ｃＤＮＡ，补平后与载体连接，转化大肠杆菌，获得阳性克隆，用３２Ｐ和光敏生物素分别标记ｃＤＮＡ制作探针，与ＣＲＳＶ及其ＲＮＡ进行点杂交，结果表明，两类不同性质的探针在检测ＲＮＡ水平上具有相同的灵敏度，其检测极限均达到１ｎｇ／ｍｌ，并对二类探针进行了比较。     

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。     

Purification of lily symptomless virus. Use and value of antisera against intact and pyrrolidine-degraded virus for testing lilies and tulips

Lily symptomless virus (LSV) was purified by clarification with chloroform, precipitation with polyethylene glycol and NaCl, and differential centrifugation. The influence of the source material and some buffers on virus yields were determined.Antisera were prepared against intact and pyrrolidine degraded LSV. It was concluded that intact and degraded LSV have very few antigenic determinants in common or none at all. The sensitivities of the micro-precipitin test and the single immunodiffusion drop test were about the same, but lower than that of electron microscopy.In the testing of lilies for LSV the most reliable results in leaves were obtained during the period from two weeks after flowering until close to the end of the growing season, and in leaves growing at a level about one-fourth of the distance from the top of the stem. In contrast to secondary infections, primary infections were detected more successfully in stored bulbs than in leaves taken from plants in the preceding growing season.In the testing of tulips, LSV was detected better in flowers than in leaves. Detection of primary infections was almost impossible. Except for those with a pink flower, experimentally infected tulips remained symptomless.Samenvatting Het symptoomloos lelievirus (LSV) were gezuiverd door klaring met chloroform, precipitatie met polyethyleenglycol en NaCl en differentieel centrifugeren. Het effect van het uitgangsmateriaal en enkele buffers op de virusopbrengst werd nagegaan.Antisera werden bereid tegen intact en met pyrrolidine afgebroken LSV. Geconcludeerd were dat intact en afgebroken LSV geen of slechts enkele antigene determinanten gemeenschappelijk hebben.De gevoeligheid van de microprecipitatietoets en de enkele immuno-diffusiedruppeltoets is ongeveer gelijk. Met het elektronenmicroscoop kunnen echter lagere virusconcentraties worden aangetoond.Het toetsen van lelies op LSV gaf de betrouwbaarste resultaten met bladeren die op ongeveer driekwart hoogte van de stengel groeien en in de periode tussen 2 weken na de bloei en vrijwel het einde van het groeiseizoen worden geplukt. Primaire infecties konden, in tegenstelling tot secundaire infecties, beter worden vastgesteld aan bolmateriaal tijdens de bewaring dan aan bladeren in het voorafgaande groeiseizoen.Bij het toetsen van tulpen werd het LSV met grotere zekerheid vastgesteld in bloemen dan in bladeren. Het vaststellen van primaire infecties was bijna niet mogelijk. Na infectie van tulpen met LSV vertoonden alleen die met een rose bloemkleur symptomen.     

Detection of two orchid viruses using quartz crystal microbalance-based DNA biosensors

ABSTRACT We have developed a piezoelectric DNA-sensor based on DNA-RNA hybridization for the detection of two orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Specific oligonucleotide probes modified with a mercaptohexyl group at the 5'-phosphate end were directly immobilized onto 10-MHz AT-cut quartz crystal microbalance (QCM). QCMs coated with such oligonucleotide probes were exposed to test solutions containing viral RNA for hybridization. Various experimental conditions evaluated were (i) DNA probe coating concentration, (ii) sensitivity and specificity of the probes at different hybridization temperatures, and (iii) effects of incubation temperature on the hybridization time. The specific nucleotide probe-coated QCM-based DNA sensors were able to detect both CymMV and ORSV in quantities as low as approximately 1 ng in purified RNA preparations and 10 ng in the crude sap of infected orchids. This is the first application of a DNA biosensor for the detection of plant viruses.     

Development of Contamination-Free Restriction Fragment Length Polymorphism Probes for the Obligate Biotroph Peronospora tabacina, an Oomycete Causing Blue Mold of Tobacco

ABSTRACT Peronospora tabacina is an obligately parasitic oomycete that causes blue mold, a devastating disease of tobacco. Genetic studies of this pathogen have been hampered by the lack of molecular markers. We generated a set of molecular markers for P. tabacina by collecting sporangiospores from infected tobacco leaves, extracting spore DNA, and cloning it in a plasmid vector. The resulting clones were then used to probe DNA from a collection of P. tabacina isolates to survey for polymorphisms. Most probes gave unexpected hybridization patterns with signal intensities that varied significantly from one DNA sample to another or between different DNA preparations of the same isolate. These results indicated that certain DNA preparations contained DNA from a source other than P. tabacina, which in turn suggested that some probes might have been derived from contaminating organisms present in the spore suspensions. Therefore, we characterized the inserts of several recombinant plasmids to determine their origins. Sequence analysis revealed that several of the inserts encoded peptides with similarity to bacterial proteins, suggesting that they were derived from bacterial contaminants. Of the remaining clones, five exhibited similarity to retroelements, one resembled eukaryotic helicase genes, and nine had no similarity to sequences in the databases. These were postulated to be true P. tabacina DNA clones. Verification of the origin of each probe was achieved by filtering a spore suspension, extracting DNA from the retentate and filtrate, and probing Southern blots of these DNA samples. These experiments confirmed the probe origins predicted by sequence analysis, resulting in the generation of 20 different restriction fragment length polymorphism probes that are specific for P. tabacina DNA. These probes should enable identification of reliable genetic markers for population studies of the blue mold organism.     

DNA probes and PCR primers for the detection of a phytoplasma associated with peanut witches'-broom

EcoRI restriction fragments of genomic DNA from the phytoplasma associated with peanut witches'-broom (PNWB) were cloned in plasmid pGEM-3Zf(+). Cloned inserts from seven PNWB-phytoplasma-specific recombinant plasmids and two subcloned plasmids were excised with restriction enzymes, labeled with digoxigenin, and used as probes. Probe PNWB281 and its derivative subclones PNWB281-4 and PNWB281-5 hybridized with DNA from PNWB-phytoplasma infected peanut and periwinkle specifically but not with DNA from healthy plants or plants infected with phytoplasmas associated with sweetpotato witches'-broom (SPWB), loofah, Ipomoea obscura, and paulownia witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Six other probes hybridized with DNA derived from PNWB and SPWB-phytoplasma-affected periwinkle but not with DNA from healthy plants or plants infected with other phytoplasmas mentioned. In Southern hybridizations, four of the nine cloned and subcloned probes could differentiate the PNWB-phytoplasma from SPWB-phytoplasma. Three primer pairs for PCR were synthesized according to the partial sequences at both ends of the cloned inserts and were able to distinguish PNWB-phytoplasma from SPWB-phytoplasma by using PCR for the first time. A minimum of 1 pg and 10 pg of total DNA from diseased periwinkle and peanut, respectively, was sufficient to amplify the specific PNWB-phytoplasma PCR fragments, allowing the detection of PNWB-phytoplasma DNA from healthy-looking periwinkle plants two weeks after graft inoculation.     

Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.     

Lily symptomless virus in tulip

Lily symptomless virus (LSV) was transmitted mechanically to only two out of 53 plant species (18 families) tested, i.e.Lilium formosanum and tulip ‘Rose Copland’ (Liliaceae). The aphid speciesMacrosiphum euphorbiae, Myzus persicae, andAphis gossypii transmitted LSV in a non-persistent manner fromLilium Mid-century hybrid ‘Enchantment’ to tulip ‘Rose Copland’.M. euphorbiae transmitted the virus more efficiently thanM. persicae andA. gossypii. The yield of LSV-infected tulips ‘Peerless Pink’ was slightly reduced compared with that of healthy tulips. LSV was observed incidentally in naturally infected commercial stocks. Two procedures to purify LSV from tulips were applied, one using crude sap and the other sap treated with chloroform. The length distribution of LSV particles in tulip showed a higher percentage of particles shorter than 600 nm as in lily.     

复合RT-PCR方法同步检测百合X病毒、百合无症病毒及百合斑驳病毒

建立了特异性检测百合X病毒(LVX)、百合无症病毒(LSV)及百合斑驳病毒(LMoV)的单一、双重及三重PCR体系并对各体系的灵敏度进行了研究。结果表明,单一PCR体系可检测的LVX最低浓度在1×10^-7μg/mL,LSV最低浓度在1×10^-8μg/mL,LMoV最低浓度在1pg/mL;双重PCR体系可明显检测的LVX、LSV最低浓度在1pg/mL,LSV、LMoV最低浓度在1ng/mL,LVX、LMoV最低浓度在1pg/mL;三重PCR体系可明显检测的LVX、LSV和LMoV的最低浓度为1ng/mL。     

A high-density random-oligonucleotide genome microarray for universal diagnostics

Microarrays offer virtually unlimited diagnostics capability, and have already been developed into diagnostic chips for many different plant pests. The full capacity of such chips, however, has lagged far behind their full potential. The main reason for this is that current chip design relies on a priori genetic information for target organisms and on a consensus on the genetic sequences to be used in particular organism groups. Such information is often unavailable and laborious to obtain. Thus, broad-application diagnostic microarrays have been limited to narrow organism groups focused on Genera of pests/pathogens or those affecting individual host crops, without applicability for simultaneous detection of diverse pests affecting many crops. This paper describes the development of a diagnostic microarray platform that has universal application based on genomic fingerprinting of any organism without a need for a priori sequence information. Taxon-specific hybridization patterns are obtained by unique hybridisation of genomic DNA to 100s–1000s of short random oligonucleotide probes. Taxon identification is then achieved by comparison of hybridisation patterns from an unknown sample against a reference-pattern database. Using bacteria as a model pathogen group, these methods deliver highly reproducible hybridisation patterns with high resolution power and enable discrimination at the species and subspecies level.     

应用细菌磁颗粒real-time RT-PCR检测百合无症病毒

为研究细菌磁颗粒分离提取不同样本材料中的植物病毒RNA的效果,以及结合实时荧光 RT-PCR技术检测LSV的灵敏性,选取感染病毒的西葫芦叶片（CGMMV和SqMV）、大豆种子（BPMV）与百合叶片（LSV和ArMV）3种样本材料,利用细菌磁颗粒分别提取这5种病毒RNA,与Trizol方法提取效果进行比较,同时与Trizol real-time RT-PCR检测LSV的灵敏度进行了比较。结果表明：BMPs方法能够从3种植物样本中提取病毒RNA,其检测LSV的灵敏性与Trizol方法相当。