太子参花叶病毒的检测与防治

1982年我们在国内外首次发现太子参花叶病毒,并对该病毒的形态结构、症状表现、宿主范围、血清学、生化特性等进行了研究,同时还对太子参病毒病进行了初步防治。在上述工作的基础上,我们又进一步应用ELISA技术,对太子参花叶病毒进行了田间检测,并确认了种子消毒方法,为太子参花叶病毒病的田间诊断与防治提出了科学措施。     

应用免疫电镜技术快速检测菜豆黄花叶病毒、马铃薯M病毒和燕麦花叶病毒

应用免疫吸附电流技术(ISEM)可有效地检测腐汁液中的菜豆黄花叶病毒(BYMV)、马铃薯M病毒(PVM)和燕麦花叶病毒(OMV)。BYMV,PVM和OMV三种抗血清的适宜工作浓度和对铜网的适宜包被时间均为1000倍和1小时,对同源病毒的适宜捕获时间分别为4℃下2、2和8小时。PVM和OMV的病汁液检测灵敏度均为稀释4000倍,而BYMV病汁液稀释16000倍时还能检测到少量病毒料子。ISEM捕获法和修饰法的结果表明,这三种病毒之间无血清学交叉反应。     

利用表面等离子体共振仪检测黄瓜花叶病毒

目的：研究一种便捷、高效地检测黄瓜花叶病毒（CMV）的方法。方法：利用表面等离子体共振（SPR）技术检测CMV。首先用11-MUA修饰SPR金片,再用EDC／NHS活化,之后通过NHS酯基与CMV抗体结合,用BSA封闭未结合的NHS酯基。将SPR金片装入SPR仪,通入待检样品,通过折射率变化实时监测实验过程。结果：该方法检测CMV的灵敏度能够达到10ng／mL,具有良好的特异性,与同属的花生矮化病毒、番茄不孕病毒无交叉反应。结论：建立的SPR方法操作简单、灵敏度高、特异性好,是一种新的高效检测CMV的方法。     

番茄花叶病毒单克隆抗体的制备及检测应用

用番茄花叶病毒(ToMV)免疫的BAL B/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗ToMV单克隆抗体(Mab)的杂交瘤细胞,其中2株能同时检测ToMV和烟草花叶病毒(TMV),各单克隆抗体腹水ELLSA效价在1∶32 000～1∶1 024 000之间。经TASELISA测定,4株单克隆抗体检测病汁液的稀释度均能达到1∶2 000倍以上。4株单克隆抗体与其他病毒无交叉反应。Westernblot分析表明,其中两株与ToMV176kD的外壳蛋白亚基有特异反应,而另两株无反应,推测它们是针对构象决定簇的抗体。     

Monoclonal Antibodies for Detection and Serotyping of Cucumber Mosaic Virus

Two monoclonal antibodies (MAbs) to cucumber mosaic virus (CMV) were selected from a panel of MAbs for use in the direct DAS (double antibody sandwich)-ELISA. Two different test procedures were developed: an ELISA with polyclonal and monoclonal antibodies (mixed ELISA) for the routine detection of CMV and a MAb-ELISA with two MAbs directed against different epitopes for the specific detection of the N serotype which is prevalent in GDR. The conventional two-step incubation of plates precoated with IgG was compared with simultaneous incubation of test sample and labelled antibody (one-step incubation). The mixed ELISA proved to be more sensitive than the direct DAS-ELISA with polyclonal antisera in detecting CMV in crude sap of infected plants. On the other hand, the MAb-ELISA could be used for serotyping of CMV isolates which is important in epidemiological investigations and in resistance breeding. Both the two-step and the one-step procedures gave similar results with some advantages of the latter procedure. One-step incubation is not only time-saving but seems to be also more sensitive with regard to the detection limit. However, care must be taken to circumvent the hook-effect occurring at high virus concentrations.     

Molecular Detection and Characterization of Chinese Yam Mild Mosaic Virus Isolates

An improved RT‐PCR was developed and validated for the detection of Yam mild mosaic virus (YMMV). Sequences of the coat protein core region of 19 Chinese isolates were obtained, and analysis indicated the presence of different genetic variants. Phylogenetic analyses showed that the Chinese isolates were divided into two distinct clusters. Complete genomic sequences of two distinct Chinese variants were determined to be 9527 and 9529 nucleotides long, excluding the 3′ poly (A) tail. Their genomic structure and organization were virtually identical to that of a Brazilian isolate. The two variants shared identity of 87.3% to one another and 83.9–84.6% to the Brazilian variant at the genomic sequence level. Phylogenetic analyses supported that they represented two distinct YMMV lineages.     

Development of a Specific Detection Technique for Cowpea Golden Mosaic Virus

Whitefly transmitted begomoviruses are on increase in tropical and subtropical countries. It is especially true of yellow mosaic viruses infecting legumes. Management of viral diseases requires specific detection techniques to demonstrate their presence in several other alternative and weed hosts. With this in view, nucleic acid hybridization test to detect Cowpea golden mosaic virus (CPGMV) in many crops and weed species was developed. This study showed that while probe to CPGMV DNA A detected all the begomoviruses, DNA B probe was highly specific and detected only CPGMV showing its specific nature.     

Purification and Characterization of an Isolate of Apple Mosaic Virus from Rose in the USA

An isolate of apple mosaic virus, designated ApMV-J, was isolated from rose cv. Sweet Surrender in New York State, USA. Mechanical transmission of ApMV-J from rose to cucumber was facilitated by preparing inocula with 0.03 M sodium phosphate buffer containing 0.02 M 2-mercap-toethanol and 1 % polyvinylpyrolidone. Catharanthus roseus cv. Little Linda was a good virus maintenance host and Cucumis sativus cv. Lemon a good propagation host. Contaminating host proteins were substantially reduced by including polyethylene glycol in the purification protocol. Purified virus had high A_260/280 nm values and contained virus particles of c 25—26 nm in diameter; most particles were isometric but a few were bacilliform. The M.Wts. of the four nucleic acids of ApMV-J, determined in non-denaturing and denaturing agarose gels, were estimated at 1.16, 0.98, 0.68 and 0.35 × 10⁶ and 1.0, 0.91, 0.66 and 0.35 × 10⁶, respectively. The viral coat protein M. Wt. was estimated to be 27 kDa.     

侵染菊花的黄瓜花叶病毒的初步鉴定和血清学检测

侵染菊花的黄瓜花叶病毒的初步鉴定和血清学检测张海保,朱西儒,张云开（中国科学院华南植物研究所广州５１０６５０）关键词菊花,黄瓜花叶病毒,间接ＥＬＩＳＡ菊花（Ｃｈｒａｙｓａｎｔｈｅｍｕｍｍｏｒｉｆｏｌｉｕｍ）病毒病是危害菊花的一类主要病害。国外文献报道...     

芜菁花叶病毒单克隆抗体的制备及检测应用

先以芜菁花叶病毒（TuMV）免疫BAL B/C小鼠,然后取其脾细胞使之与SP2/0鼠骨髓瘤细胞融合,经筛选、克隆,获得4株能稳定传代并分泌抗TuMV单克隆抗体（Mab）的杂交瘤细胞,并以之制备腹水单抗。4株单克隆抗体腹水ELISA效价在10-5～10-6之间,仅对TuMV起特异性反应。Western blot分析表明,4株单抗都能与TuMV 34kD的外壳蛋白亚基起特异反应。利用TuMV的多抗兔血清和单抗腹水建立了三抗体夹心ELISA检测TuMV的方法,检测病叶的灵敏度为1∶5120倍,检测提纯TuMV病毒绝对量为21.9 ng。利用三抗体夹心ELISA测定出7种作物上有TuMV侵染。      

由高粱花叶病毒和甘蔗花叶病毒引发的浙江甘蔗花叶病害

从浙江省5个地区采集表现花叶症状的甘蔗病叶,用马铃薯Y病毒科简并引物做PCR扩增及测序鉴定.序列分析表明,5个甘蔗样品均含有高粱花叶病毒(SrMV),其中3个样品中还存在甘蔗花叶病毒(SCMV)的复合侵染.序列比较和系统进化树分析表明,浙江甘蔗样品中的SrMV序列彼此很相似,核苷酸同源性大于93%,与已报道的4个美国分离物在CP区域同源性很高,但是3′非编码区的同源性却仅为70%左右.SCMV欧洲和中国玉米分离物及美国、南非和澳大利亚甘蔗分离物分别形成两个远缘群体,浙江甘蔗分离物群体位于两者之间;群体间CP氨基酸序列同源性均大于80%.甘蔗和玉米上的SCMV差异明显,多为无义突变.     

香蕉试管苗黄瓜花叶病毒检疫检验技术研究

建立了香蕉试管苗黄瓜花叶病毒ＤＡＣ－ＥＬＩＳＡ检测技术。应用０．１ｍｏｌ／Ｌ磷酸盐缓冲液（ｐＨ７．２,内含１％ＮａＤＩＥＣＡ）作为样品制备缓冲液,检测灵敏度可达到１／５１２０（稀释度）。两年来检测了１２８７个样品,香蕉试管苗生产繁殖材料的带毒率为２．８％。     

The Detection of the Abutilon Mosaic Virus Depends on the Experimental Daytime

Fluorescence and electron microscopical studies show that the detection of structures associated with the Abutilon Mosaic Virus disease is dependent on the sampling daytime. Moreover the infectivity of plant sap may vary during the day. The results emphasize the importance to consider the daytime as an essential parameter in experimental design in virological research.     

Sequence Analysis and Detection Using Immunocapture-PCR of Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus in Hawaiian Orchids

A Hawaiian isolate of Cymbidium mosaic virus (CyMV-H) was purified from Dendrobium orchid, and a cDNA library was constructed. Clones containing the coat protein (CP) gene and movement protein (MP) gene were identified by colony hybridization and polymerase chain reaction (PCR). The Hawaiian isolate of Odontoglossum ringspot virus (ORSVH) was purified from Cattleya orchid. The CyMV CP gene was PCR amplified from a cDNA done. The ORSV CP and 54 kDa putative replicase genes and CyMV-MP gene were cloned by RT-PCR Sequences of these genes of CyMV-H and ORSV-H were compared with those of CyMV and ORSV from Singapore, Japan. Korea, and Germany. The high degree of sequence identity (91–99%) at the nucleotide level for all gene sequences analysed, shows that CyMV and ORSV from different countries are closely related. Sequence comparison results show that CyMV strains can be divided into two groups based on differences in amino acid sequences of the coat protein gene: CyMV-H closely resembles CyMV-SI while CyMV-S2 resembles CyMV-K, A sensitive, rapid, and reliable immunocapture PCR (ICPCR) assay was developed to detect both viruses, CyMV was detected from dilutions equivalent to 100 mg of orchid material and ORSV was detected from dilutions equivalent to 10 μg of orchid material. IC-PCR was compared with direct binding PCR (DB-PCR) and ELISA for their sensitivities.     

Elimination of Apple Mosaic Virus and Raspberry Bushy Dwarf Virus from Infected Red Raspberry (Rubus idaeus L.) by Tissue Culture

Experiments with ApMV infected ‘Malling Landmark’ and RBDV infected ‘Schamp;önemannamp;’ and ‘Trentamp;’ plants were carried out to evaluate a) the dependence of virus eradication on explant size and mass propagation. b) the reliability of results of ELISA tests on in vitro plantlets. With ApMV a correlation between virus elimination and explant size was observed, whereas with RBDV even plantlets from the smallest established explants were still infected. With ApMV, in vitro multiplication for three subsequent subcultures did not lead to further virus elimination, with RBDV this was observed in two cases. ELISA test results for both viruses, ApMV and RBDV, were identical when small in vitro plantlets, long-term stored plants, or potted plants from the same origin were tested, indicating that virus tests are possible with very young plant material and can be used to select virus-free plants in vitro. Tissue culture permits long-term storage of plant viruses. It is also suitable tor plant virus propagation and could be a useful aid in plant virus purification. For commercial multiplication only virus-indexed plant material should be used for establishment and further propagation in vitro.