Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon,Salmo salar,infected with salmonid alphavirus (SAV3)          下载免费PDF全文

M Braceland  J Tinsley  D Cockerill  R Bickerdike  M F McLoughlin  P D Eckersall 《Journal of fish diseases》2017,40(8):1077-1087

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on‐farm qualitative test and an in‐laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ₃₄₀). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ₃₄₀ in infected fish rose from undetectable to a maximum at 6 weeks post‐infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.  相似文献   

A PCR-based 'molecular tool box' for in planta differential detection of Verticillium dahliae vegetative compatibility groups infecting artichoke     

M. Collado-Romero  M. Berbegal  R. M. Jiménez-Díaz  J. Armengol  J. Mercado-Blanco 《Plant pathology》2009,58(3):515-526

A multiplex-nested-PCR procedure was developed for in planta detection of Verticillium dahliae isolates infecting artichoke and assessment of their vegetative compatibility groups (VCGs). PCR markers were identified and assigned to V. dahliae VCGs, including: i) a 334 bp marker amplified from VCG1A or VCG2B³³⁴ isolates; ii) a 688 bp marker amplified from VCG2A or VCG4B isolates; and iii) a 688 bp and a 964 bp PCR marker amplified from VCG2B⁸²⁴ isolates. The infecting V. dahliae VCGs were identified in artichoke tissues according to specific patterns of amplified markers after two rounds of PCR. The PCR-based 'molecular tool box' was first optimized using DNA extracted from artichoke plants artificially inoculated with isolates representative of known VCGs. Thereafter, the efficiency of the molecular procedure was tested using DNA extracted from naturally-infected artichoke plants showing a range of symptom severity as well as from symptomless plants. The novel multiplex-nested-PCR assay was clearly superior in detecting the pathogen compared to conventional isolation procedures, and in addition was informative about the VCGs. Moreover, the PCR method allowed the detection and VCG identification of V. dahliae infections in symptomless but infected plants, which had yielded false negatives when checked by microbiological isolation procedures. This 'molecular tool box' has uncovered the presence of several V. dahliae VCGs infecting the same artichoke plants in the Comunidad Valenciana Region. In addition, it is useful for genetic and pathogenicity diversity studies of V. dahliae populations infecting artichoke, and may help in predicting the severity of verticillium wilt epidemics.  相似文献   

Detection and quantification of airborne inoculum of Sclerotinia sclerotiorum using quantitative PCR     

S. L. Rogers  S. D. Atkins  J. S. West 《Plant pathology》2009,58(2):324-331

This paper reports the development of a new specific diagnostic technique to accurately quantify airborne inoculum of Sclerotinia sclerotiorum and discusses its potential use in disease-forecasting schemes, using examples of three contrasting epidemic seasons: 2007, when there was a severe epidemic of sclerotinia stem rot (SSR) in England and high numbers of airborne ascospores were trapped at Rothamsted, and, in contrast, 2003 and 2004, when the incidence of SSR in England was low and low numbers of airborne ascospores were trapped at Rothamsted. DNA was extracted from wax-coated plastic tapes, such as those used in Burkard (Hirst-type) spore traps and rotating-arm traps. A SYBR-green quantitative PCR (qPCR) method produced a linear relationship between ascospore numbers and S. sclerotiorum DNA (mean 0·35 pg DNA per spore) and was able to detect DNA representing as few as two ascospores. The technique was insensitive to DNA of the host plant, Brassica napus , and other plant pathogens, including Sclerotinia minor , S. trifoliorum and Botrytis cinerea , and common airborne fungal genera such as Cladosporium and Penicillium . There was no relationship between rainfall and numbers of airborne ascospores of S. sclerotiorum present at Rothamsted during the period of infection in the severe SSR season (2007).  相似文献   

Geographical distribution and molecular insights into abamectin and milbemectin cross‐resistance in European field populations of Tetranychus urticae     

Wenxin Xue  Simon Snoeck  Christine Njiru  Emre Inak  Wannes Dermauw  Thomas Van Leeuwen 《Pest management science》2020,76(8):2569-2581

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信息技术支持下的中医诊断学“自主学习”模式构建与实践     

孙贵香  黄惠勇  刘伟  倪佳  袁肇凯  简维雄  李琳   《勤云标准版测试》2014,34(8):56-59

探讨信息技术支持下的中医诊断学"自主学习"模式的概念、构建方法和思路,并以舌诊教学为例,介绍实施的具体方法。为实践"以学生为本"的教育理念,推动中医诊断学教学改革做出了有益的尝试。 更多还原  相似文献   

Molecular identification of iridoviruses infecting various sturgeon species in Europe          下载免费PDF全文

L Bigarré  M Lesne  A Lautraite  V Chesneau  A Leroux  M Jamin  P M Boitard  A Toffan  M Prearo  S Labrut  P Daniel 《Journal of fish diseases》2017,40(1):105-118

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low‐to‐high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74–88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus‐European (AcIV‐E). Moreover, three samples infected with AcIV‐E showed genetic heterogeneity, with the co‐existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV‐E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV‐E and an NV‐related virus.  相似文献   

Identification and differentiation of Phyllosticta species causing freckle disease of banana using high resolution melting (HRM) analysis     

M.‐H. Wong  J. Henderson  A. Drenth 《Plant pathology》2013,62(6):1285-1293

Freckle disease of banana is caused by three closely related species of Phyllosticta, namely P. musarum, P. maculata and P. cavendishii. In this study, a high resolution melting (HRM) analysis assay was developed and its potential to identify these three fungal species is reported. The assay, which targets the ITS of the nuclear rDNA of the fungal species, generates three distinct melt profiles for the three Phyllosticta species. It is also able to distinguish a combination of up to three co‐infecting species by generating a deviant melt curve. Thirty‐five fungal cultures and infected herbarium leaf specimens, previously characterized using nucleotide sequencing as belonging to one of the three Phyllosticta species, were used for validation of the HRM analysis assay. The normalized curves generated differentiated all samples, with samples from each species correctly identified. The assay was further evaluated against 18 uncharacterized infected leaf specimens from various geographic locations and the results were verified by subsequent nucleotide sequencing. This HRM analysis assay allows rapid identification and differentiation of the three Phyllosticta species using a single primer pair in a one‐step closed‐tube system without labelled fluorescence probes. This novel assay format has potential for simultaneously identifying and differentiating other closely related species of plant pathogens, as well as the classification of infected historic specimens.  相似文献   

Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences     

L. Pritchard  S. Humphris  G. S. Saddler  N. M. Parkinson  V. Bertrand  J. G. Elphinstone  I. K. Toth 《Plant pathology》2013,62(3):587-596

This study used a novel computational pipeline to exploit draft bacterial genome sequences in order to predict, automatically and rapidly, PCR primer sets for Dickeya spp. that were unbiased in terms of diagnostic gene choice. This pipeline was applied to 16 draft and four complete Dickeya genome sequences to generate >700 primer sets predicted to discriminate between Dickeya at the species level. Predicted diagnostic primer sets for both D. dianthicola (DIA‐A and DIA‐B) and ‘D. solani’ (SOL‐C and SOL‐D) were validated against a panel of 70 Dickeya reference strains, representative of the known diversity of this genus, to confirm primer specificity. The classification of the four previously sequenced strains was re‐examined and evidence of possible misclassification of three of these strains is presented.  相似文献   

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species     

S. L. van Brunschot  C. F. Gambley  P. J. De Barro  R. Grams  J. E. Thomas  J. Henderson  A. Drenth  A. D. W. Geering 《Plant pathology》2013,62(5):1132-1146

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.  相似文献   

Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans,its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis     

《Soil Science and Plant Nutrition》2013,59(4):478-484

Abstract

A preliminary survey using 20 conventionally farmed fields in which fumigants have been applied every year showed that the root-lesion nematode Pratylenchus penetrans was distributed both in the upper (0–30?cm) and lower (30–60?cm) soil layers. In six of the 20 fields, P.?penetrans was detected in the lower layers exclusively, suggesting that the most appropriate depth to sample soil is 0–60?cm to estimate the relationship between the density of P.?penetrans and its damage to radish. There was a highly significant correlation (r?=?0.923) between the density of P.?penetrans in the 0–60?cm depth and the number of spots on a radish. No damage to radish was observed in soils with <2.5 individuals of P.?penetrans per 20?g soil before cultivation. However, in cases in which the density of P.?penetrans was 3.4–6.2 individuals per 20?g soil, the number of spots on a radish showed more variation (0–131.5 per radish) and there was no significant correlation between them. The nematode community structure of soils with 3.4–8 individuals of P.?penetrans per 20?g soil, evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis, was significantly different (anova, PC2, P?<?0.05) between soils with low (0–42) and high (more than 80) damage levels, suggesting that radish damage might be predicted on the basis of the prevailing soil nematode community structure.  相似文献