Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR   总被引：4，自引：1，他引：4  

D. W. Cullen  A. K. Lees  I. K. Toth  J. M. Duncan † 《Plant pathology》2002,51(3):281-292

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .  相似文献   

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>     

Judith Hübschen  Lilo Kling  Ulrike Ipach  Volker Zinkernagel  Nathalie Bosselut  Daniel Esmenjaud  Derek J.F. Brown  Roy Neilson 《European journal of plant pathology / European Foundation for Plant Pathology》2004,110(8):779-788

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.  相似文献   

A framework for evaluation of on-farm mastitis diagnostics in Australia     

RN Zadoks  E Scholz  SM Rowe  JM Norris  HB Pooley  J House 《Australian veterinary journal》2023,101(4):142-152

Numerous culture-based diagnostics are available on the Australian and international markets for on-farm detection of bacterial pathogens in milk. Use of such diagnostics may provide an opportunity to improve the prudent use of antimicrobials in udder health management. Farms are low-resource settings in terms of diagnostic microbiology capacity. The World Health Organisation has identified criteria for the evaluation of diagnostic tests in low resource settings based on Accuracy, Sensitivity, Specificity, User-friendliness, being Rapid or Robust, Equipment-free and being Deliverable (ASSURED). Here, we review how those criteria can be interpreted in the context of microbiological diagnosis of mastitis pathogens, and how on-farm diagnostics that are currently available in Australia perform relative to ASSURED criteria. This evaluation identifies multiple trade-offs, both with regard to scientific criteria and with regards to convenience criteria. More importantly, the purpose of testing may differ between farms, and test performance should be evaluated relative to its intended use. The ability of on-farm mastitis diagnostics to inform mastitis treatment decision-making in a timely and cost-effective manner depends not just on test characteristics but also on farm-specific pathogen prevalence, and on the farm enterprise's priorities and the farm manager's potential courses of action. With most assay evaluations to date conducted in professional laboratories, there is a surprising dearth of information on how well any of the diagnostic tests perform on-farm and, indeed, of the on-farm decision-making processes that they aim to inform.  相似文献   

Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics     

M. Hren  J. Boben  A. Rotter  P. Kralj  K. Gruden  M. Ravnikar 《Plant pathology》2007,56(5):785-796

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   

Use of random amplified polymorphic DNA (RAPD) to identify races 1, 2, 4 and 8 of Fusarium oxysporum f. sp. dianthi in Italy     

Quirico Migheli  Elena Briatore  Angelo Garibaldi 《European journal of plant pathology / European Foundation for Plant Pathology》1998,104(1):49-57

The RAPD fingerprinting procedure was used in combination with pathogenicity assays on differential cultivars to characterize a representative collection of 72 Fusarium spp. isolates of different geographic origin collected from diseased carnation. In F. oxysporum f. sp. dianthi, isolates were grouped according to the physiologic race: group 1 included isolates of race 4; group 2 was formed by isolates of race 2 and single representatives of races 5 and 6; group 3 included isolates of races 1 and 8. No correlation was found between RAPD data and geographic origin of the isolates tested: representatives of race 2 isolated in Italy, Israel and Japan had the same amplification profile. Three isolates which showed a low level of pathogenicity on all carnation cultivars tested shared an identical amplification pattern and are probably saprophytic F. oxysporum. Finally, two F. redolens isolates from Japan and seven non-pathogenic isolates of F. proliferatum collected from diseased carnation in Italy, Israel and The Netherlands were clearly distinguishable according to their RAPD fingerprint. The results are discussed in relation to previous studies on the genetic diversity of F. oxysporum f. sp. dianthi and to the development of forma specialis- and pathotype-specific diagnostic tools.  相似文献   

激光诊断技术在内燃机研究中的应用及其分析     

赵昌普 《拖拉机与农用运输车》2009,36(6):1-5

论述了在内燃机缸内速度测量、喷雾粒子粒度测量及燃烧过程温度和组分浓度测量中常用的激光诊断技术和测试方法,并对其工作原理、适用范围与场合、优缺点等进行了比较和分析。相位多谱勒粒子测速(PDPA)技术比较适合于单点速度、粒子尺寸及其分布的测量;粒子图像测速(PIV)技术适合于缸内二维平面速度场的测量;激光全息术是分析喷雾特性的有效途径和测量喷雾液滴尺寸的标准方法;平面激光诱导荧光(PLIF)法已成为喷雾、燃烧过程组分浓度及火焰结构研究的重要工具。  相似文献   

A practitioner's guide to understanding equine infectious disease diagnostics in the United Kingdom. Part 1: How to optimise sampling approaches and a guide to agent detection testing methods     

F. M. Whitlock  J. R. Newton 《Equine Veterinary Education》2022,34(6):330-336

Diagnostic testing is routinely performed by the equine clinician when dealing with suspected infectious disease cases and outbreaks. Optimal sample timing, choice and handling are fundamental to attain an accurate diagnosis, and a good understanding of laboratory-based sample analysis techniques, and their validation is necessary for effective diagnostic test result interpretation. This two-part series highlights the importance of interpreting results bearing testing limitations and specific clinical findings in mind, and on these foundations, the treating clinician should always be well placed to deal with equine infectious diseases. Part 1 in this series will provide a treating clinician with an overview of the importance of testing horses in infectious disease outbreaks and how this is achieved. The different laboratory testing options available for agent detection and their methods will also be discussed. Part 2 will summarise serological (antibody) testing techniques, sample processing (including how tests are performed and validated) and result interpretation.  相似文献   

The potential of biotechnology in temperate agroforestry practices     

N. B. Klopfenstein  J. G. Kerl 《Agroforestry Systems》1995,32(1):29-44

Technologies in forest molecular biology and tissue culture could play an increasing role in the choice of genotypes for successful establishment of agroforestry practices. Research areas such as micropropagation, somatic embryogenesis, genetic engineering, marker-aided selection, and molecular diagnostics are merging with traditional forest biological studies to help identify and produce better-suited trees for agroforestry plantings. A combination of classical and molecular biological research could be used to improve pest and stress resistance of selected genotypes, modify structure and function, and monitor pests of trees. This merger of approaches, as well as continued technological development, could accelerate the production and selection of suitable tree genotypes for agroforestry plantings.The US Government right to retain a non-exclusive, royalty free licence in and to any copy-right is acknowledged.  相似文献   

A diagnostic approach to the pruritic horse          下载免费PDF全文

S. D. White 《Equine Veterinary Education》2015,27(3):156-166

Pruritus is a common complaint in equine medicine. While pruritus in most horses is due to ectoparasites (midges, flies, mites) or environmental allergens (pollens, barn dust, moulds, etc.), other causes such as staphylococcal or fungal infections, vasculitis, and internal organ dysfunction should not be overlooked. In a rational approach to the pruritic horse, history and physical examination become very important as a guide to choosing diagnostic tests.  相似文献   

An oligonucleotide microarray-based assay for identification of phytoplasma 16S ribosomal groups     

M. Nicolaisen  A. Bertaccini 《Plant pathology》2007,56(2):332-336

A method using consensus PCR followed by oligonucleotide microarray hybridization was developed for identification of phytoplasma 16Sr ribosomal groups. The array consisted of 21– to 33-nt-long oligonucleotides which were designed to hybridize to individual 16Sr groups. Two oligonucleotides were designed to detect all phytoplasma groups. The array could efficiently identify samples from 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, 16SrVII, 16SrIX, 16SrX and 16SrXII ribosomal groups. This microarray-based test represents a rapid method for detection of phytoplasmas in unknown samples and for identification of most 16Sr groups.  相似文献