共查询到20条相似文献，搜索用时 15 毫秒
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T. CANDRESSE G. MACQUAIRE M. LANNEAU M. BOUSALEM T. WETZE L. QUIOT-DOUINE J. B. QUIOT J. DUNEZ 《EPPO Bulletin》1994,24(3):585-594
We have developed an immunocapture-PCR (IC-PCR) detection technique for plum pox potyvirus (PPV) which is both simple and highly sensitive. This single-day assay can detect about 2000 virus particles (200 fg of virus) diluted in 100 μl of crude plant sap, which is equivalent to a sensitivity about 2000 times better than that of a standard ELISA assay. RFLP analysis and sequencing of the amplified cDNA fragment indicate that three groups of strains with limited intra-group variability can be discriminated. Two of these groups correspond to the previously described D and M serotypes of PPV. The third group contains, so far, only the El Amar Egyptian isolate. Strains belonging to the D or M serotypes can easily be discriminated by Rsal polymorphism in the amplified cDNA fragment. Synthetic oligonucleotides allowing specific amplification of PPV strains belonging either to the D or to the M serotypes have also be designed. 相似文献
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A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY. 相似文献
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K.-H. Pastrik 《European journal of plant pathology / European Foundation for Plant Pathology》2000,106(2):155-165
A new multiplex PCR assay was developed for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers. The assay combines two different tests in one reaction mixture. First, a highly specific and sensitive detection of the pathogen and second, an indicator test for successful amplification (internal PCR control), which monitors potentially false-negative PCR results, caused by inhibition of the PCR. For the simultaneous amplification of two different targets in one reaction mixture, a mix of two different primer sets was used. For the detection of C. michiganensis subsp. sepedonicus, a pathogen-specific primer set PSA-1/PSA-R was used, based on the intergenic spacer region of the 16S–23S rRNA genes of C. michiganensis subsp. sepedonicus. For the simultaneous amplification of the internal PCR control, the plant-specific primer set NS-7-F/NS-8-R was employed, permitting amplification of target sequence from plant DNA present in DNA extractions from potato core fluid. The applicability of the multiplex PCR was verified in 3500 composite samples of 200 seed potato tubers from 143 different cultivars in a survey for C. michiganensis subsp. sepedonicus by parallel testing using immunofluorescence, a bioassay in eggplant seedlings and multiplex PCR. 相似文献
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A disease caused by potato Y potyvirus (PVY) affects tomato plantations with variable severity in Tenerife Island. Affected plants show diverse symptoms such as necrotic lesions or mild to severe mosaic in leaves and whitish spots in green fruits that remain after ripening. Tomato PVY isolates and few potato and capsicum PVY isolates have been characterized on the basis of biological, serological and molecular criteria. All PVY isolates reacted positively to monoclonal antibodies specific for PVYO/C or PVYN strains, and nearly 50% of tomato PVY isolates were recognized by both. Differentiation of PVY strains according to the response of inoculated experimental plants was confusing due to the variability of viral aggressiveness and symptomatology induced. RFLP analysis of the CP gene and 3’untranslated region (UTR) revealed high variability. In addition to mixed infection by different PVY strains, the biological and molecular properties of those tomato PVY isolates that react to both monoclonal antibodies could be explained as the result of RNA recombination between distinct PVY strains which infect the same host plant. 相似文献
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A potyvirus (eggplant mottle virus, EMoV) causing mosaic mottling in eggplant ( Solanum melongena ) was characterized on the basis of biological, serological and partial nucleotide sequence properties. EMoV infected Chenopodium amaranticolor and members of the Solanaceae. Polyclonal antiserum against EMoV showed antigenic relationship with henbane mosaic potyvirus (HMV) and potato Y potyvirus (PVY). Virus-specific antibodies directed to the N-terminal region of EMoV cross-reacted only with PVY. Determination and comparison of nucleotide sequence of the coat protein (CP) and the 3'-untranslated region (UTR) of EMoV with other potyviruses showed that the level of homology was highest with PVY isolates. Comparative sequence analyses of the CP amino acid and 3'-UTR sequences with distinct PVY isolates placed EMoV within the PVYO subgroup. 相似文献
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A diagnostic tool for improved detection of Xanthomonas fragariae using a rapid and highly specific LAMP assay designed with comparative genomics 
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下载免费PDF全文 M. Gétaz A. Bühlmann P. H. H. Schneeberger C. Van Malderghem B. Duffy M. Maes J. F. Pothier B. Cottyn 《Plant pathology》2017,66(7):1094-1102
Molecular diagnostics of plant pathogens are crucial to prevent disease spread and to enhance food quality and security. A comparative genomics approach using genomes of different Xanthomonas species and pathovars was applied to identify highly specific targets in the genome of Xanthomonas fragariae, the causal agent of angular leaf spot of strawberry, listed under quarantine regulations in Europe. A reliable and sensitive loop‐mediated isothermal amplification (LAMP) assay was designed using a unique marker, providing a highly specific and rapid detection technique, convenient for on‐site detection. Specificity of the designed assay was tested on 37 strains from a culture collection of X. fragariae, 82 strains of other Xanthomonas species and pathovars and 11 strains of other bacterial genera isolated from strawberry leaves. A detection limit of 102 fg was achieved, approximating to 20 genome copies per reaction. When performing analyses with crude plant material, a consistent lower detection efficiency of 102 CFU mL?1 was achieved. The LAMP assay designed in this study was adapted to work on crude plant material without any prior extensive extraction steps or incubation period; moreover, it does not require advanced analytical knowledge or a fully equipped laboratory. Results were produced within 7–20 min, depending on the pathogen concentration, thus providing a high‐throughput and user‐friendly method for detection and screening of plant material in support of quarantine regulations. 相似文献
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A novel pair of universal primers was developed to detect potyvirus species after conserved sites were identified using all full‐length potyvirus sequences available by 2005. The breadth of specificity of the new primers, NIb2F and NIb3R, was investigated and compared with the specificity of two routinely used primer pairs in plant virus diagnostic laboratories. RNA from 40 potyvirus isolates representing 23 recognized and three possible new species was tested. Reactions with NIb2F and NIb3R produced amplicons of 350 bp from all 40 virus isolates tested. Reactions with the previously published WCIEN and Potyvirid primers amplified cDNA from 32 and 21 isolates, representing possibly 21 and 15 species, respectively. The identity of 12 unknown potyvirus isolates was confirmed by sequencing and three were found to be potentially distinct potyvirus species. Gel banding patterns from reactions with NIb2F and NIb3R were simpler to interpret than those from reactions with the other two primer sets; fewer products were visible and the cDNA fragments were less variable in size. RT‐PCR with the novel primers is predicted to be able to detect virus isolates from all major groups within the genus Potyvirus and its reliability makes it well suited for use as a routine diagnostic assay. 相似文献
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Candresse T Cambra M Dallot S Lanneau M Asensio M Gorris MT Revers F Macquaire G Olmos A Boscia D Quiot JB Dunez J 《Phytopathology》1998,88(3):198-204
ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates. 相似文献
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M. RAVELONANDRO R. SCORZA A. CALLAHAN J. CORDTS M. MONSION M. FUCHS R. DELBOS J. BACHELIER D. GONSALVES J. DUNEZ 《EPPO Bulletin》1994,24(3):713-719
Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R2 progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums. 相似文献
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PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity
and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation
(HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a
472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption
and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from
soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps.
A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved
co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed
well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction
methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity
assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according
to EPPO guidelines. 相似文献
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A very sensitive and simple procedure has been developed for the preparation of plant samples before PCR amplification using direct tissue printing. This technique was successfully used for the detection of plum pox potyvirus, apple chlorotic leaf spot trichovirus, prunus necrotic ringspot ilarvirus and apple mosaic ilarvirus, in different stone-fruit material (peach, apricot, plum and almond). The results demonstrate the general applicability of this technique. 相似文献
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香蕉条斑病毒LAMP快速检测方法的建立 总被引：1，自引：0，他引：1
环介导等温扩增（loop-mediated isothermal amplification,LAMP）是一种特异、灵敏、快速的新型基因检测技术。本研究以香蕉条斑病毒（Banana streak virus,BSV）ORF3保守区域为基础针对6个特定区域设计并筛选了4条LAMP扩增引物,通过对LAMP反应中MgSO4、dNTPs、Betaine等主要试剂浓度进行优化,建立了香蕉BSV的LAMP检测方法,63℃反应90 min后通过在反应产物中添加SYBR Green Ⅰ染料后颜色的变化,肉眼即可判断检测结果。LAMP具有极高的检测特异性和灵敏性,其检测下限约为3.2 ng·μL-1,是PCR检测灵敏度的25倍,能快速、准确地对疑似样品进行检测,本研究对华南地区部分疑似样品的检测结果显示LAMP阳性检出率比PCR检出率高。本文建立的BSV LAMP检测方法是对BSV检测方法的拓展和延伸,为香蕉病毒的快速检测提供技术保障。 相似文献
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ABSTRACT Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts. 相似文献
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二重RT-PCR快速检测马铃薯病毒的方法 总被引：6，自引：0，他引：6
本研究采用传统的蛋白酶K法和病毒RNA简易浸提法,从马铃薯块茎、茎干、叶梗、叶片中提取马铃薯X病毒,马铃薯Y病毒,马铃薯A病毒及马铃薯卷叶病毒RNA,并设计了4种马铃薯病毒引物,优化了二重RT-PCR反应条件,可以同步扩增出上述4种病毒,扩增产生的靶带分别为562bp(PVX)、480bp(PVY)、336bp(PLRV)、255bp(PVA).应用病毒RNA简易制样技术和优化的二重RT-PCR反应条件,可以同步快速检测田间自然感染的马铃薯病毒,此研究还可适合于检测马铃薯脱毒种薯及试管苗,对马铃薯病毒病早期监测有一定的作用. 相似文献