Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification: Testing for Specificity Using a Standardized Library of Isolates

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as β-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

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Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue

Phytopathology ◽

10.1094/phyto-05-14-0134-r ◽

2015 ◽

Vol 105 (2) ◽

pp. 265-278 ◽

Cited By ~ 49

Author(s):

Timothy D. Miles ◽

Frank N. Martin ◽

Michael D. Coffey

Keyword(s):

Real Time ◽

Internal Control ◽

Plant Tissue ◽

Pathogen Detection ◽

Phytophthora Ramorum ◽

Isothermal Amplification ◽

Sample Collection ◽

Phytophthora Spp ◽

Wide Range ◽

Tissue Maceration

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

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Adaptation of a Phytophthora ramorum Real-Time Polymerase Chain Reaction Assay Based on a Mitochondrial Gene Region for Use on the Cepheid SmartCycler

Plant Health Progress ◽

10.1094/php-2010-0212-01-rs ◽

2010 ◽

Vol 11 (1) ◽

pp. 28

Author(s):

K. E. Sechler ◽

M. M. Carras ◽

N. Shishkoff ◽

P. W. Tooley

Keyword(s):

Real Time ◽

Mitochondrial Gene ◽

Cross Reactivity ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Rt Pcr ◽

Field Samples ◽

Symptomatic Tissue ◽

Polymerase Chain ◽

High Degree

Detection of Phytophthora ramorum in US commercial nurseries has led to a number of quarantine regulations. Methods such as real-time PCR (RT-PCR) provide rapid and reliable detection that can supplement attempts to culture P. ramorum from symptomatic tissue. We adapted and optimized a previously described mitochondrial gene-based RT-PCR assay for use with a Cepheid SmartCycler v.1 and ready-to-use lyophilized PCR beads. The detection limit was 10 fg of P. ramorum genomic DNA. No cross-reactivity was observed on the SmartCycler for seven additional Phytophthora species tested, which included species known to cross-react in other assays as well as recently described species Phytophthora foliorum and P. kernoviae. The SmartCycler assay described here was used to detect P. ramorum in a set of 2008 California field samples with a high degree of accuracy. Accepted for publication 13 October 2009. Published 13 February 2010.

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Development of an Efficient Real-Time Quantitative PCR Protocol for Detection ofXanthomonas arboricolapv. pruni inPrunusSpecies

Applied and Environmental Microbiology ◽

10.1128/aem.01593-10 ◽

2010 ◽

Vol 77 (1) ◽

pp. 89-97 ◽

Cited By ~ 33

Author(s):

Ana Palacio-Bielsa ◽

Jaime Cubero ◽

Miguel A. Cambra ◽

Raquel Collados ◽

Isabel M. Berruete ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Plant Material ◽

Pathogen Detection ◽

Plant Protection ◽

The European Union ◽

Real Time Quantitative Pcr ◽

Field Samples ◽

Mediterranean Plant ◽

Different Origins

ABSTRACTXanthomonas arboricolapv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as beingX. arboricolapv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system inX. arboricolapv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 102CFU ml−1, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed forX. arboricolapv. pruni strains from different origins as well as for closely relatedXanthomonasspecies, non-Xanthomonasspecies, saprophytic bacteria, and healthyPrunussamples. The efficiency of the developed protocol was evaluated with field samples of 14Prunusspecies and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, andX. arboricolapv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test forX. arboricolapv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

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Development of a Recombinase Polymerase Amplification Fluorescence Assay for the Detection of Canine Adenovirus 2

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.596877 ◽

2021 ◽

Vol 8 ◽

Author(s):

Li Xiao ◽

Mengdi Zhang ◽

Zhige Tian ◽

Ye Ge ◽

Tongyuan Zhang ◽

...

Keyword(s):

Real Time ◽

Canine Distemper Virus ◽

Pathogen Detection ◽

Detection Method ◽

Adenovirus Type ◽

Recombinase Polymerase Amplification ◽

Clinical Samples ◽

Clinical Applicability ◽

Field Samples

Canine adenovirus type 2 (CAdV-2) is often found in co-infections with other pathogens causing canine infectious respiratory disease (CIRD). Rapid, efficient, and convenient pathogen detection is the best approach for early confirmatory diagnosis. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) assay for detection of canine adenovirus 2 (CAV), which can detect CAV within 15 min at 39°C. The detection limit that assay was 214 copies/μl DNA molecules per reaction. The specificity was indicated by a lack of cross-reaction with canine distemper virus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV). Field and clinical applicability of this assay were evaluated using 86 field samples. The coincidence rate of the detection results for clinical samples between CAV-RPA and qPCR was 97.7%. In summary, the real-time CAV-RPA analysis provides an efficient, rapid and sensitive detection method for CAV.

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Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

Phytopathology ◽

10.1094/phyto.2004.94.10.1075 ◽

2004 ◽

Vol 94 (10) ◽

pp. 1075-1083 ◽

Cited By ~ 101

Author(s):

Katherine J. Hayden ◽

David Rizzo ◽

Justin Tse ◽

Matteo Garbelotto

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Pathogen Detection ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Plant Diseases ◽

Chain Reaction ◽

Nested Polymerase Chain Reaction ◽

New Host ◽

Polymerase Chain

The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

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Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

Phytopathology ◽

10.1094/phyto-97-5-0632 ◽

2007 ◽

Vol 97 (5) ◽

pp. 632-642 ◽

Cited By ~ 70

Author(s):

G. J. Bilodeau ◽

C. A. Lévesque ◽

A. W. A. M. de Cock ◽

C. Duchaine ◽

S. Brière ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Severe Disease ◽

Phytophthora Ramorum ◽

Sybr Green ◽

Molecular Beacons ◽

Positive Real ◽

Phytophthora Spp ◽

Pcr Assays ◽

Β Tubulin

Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), β-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using β-tubulin and seemed to be more sensitive.

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Membrane-Based Oligonucleotide Array Developed from Multiple Markers for the Detection of Many Phytophthora Species

Phytopathology ◽

10.1094/phyto-04-12-0092-r ◽

2013 ◽

Vol 103 (1) ◽

pp. 43-54 ◽

Cited By ~ 17

Author(s):

Wen Chen ◽

Zeinab Robleh Djama ◽

Michael D. Coffey ◽

Frank N. Martin ◽

Guillaume J. Bilodeau ◽

...

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Plant Pathogens ◽

Sequence Data ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Oligonucleotide Array ◽

Data Set ◽

Phytophthora Spp ◽

Field Samples

Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5′ end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

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Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

Phytopathology ◽

10.1094/phyto-96-0336 ◽

2006 ◽

Vol 96 (4) ◽

pp. 336-345 ◽

Cited By ~ 60

Author(s):

Paul W. Tooley ◽

Frank N. Martin ◽

Marie M. Carras ◽

Reid D. Frederick

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Limit Of Detection ◽

Multiplex Assay ◽

Phytophthora Ramorum ◽

Chain Reaction ◽

Plant Dna ◽

Field Samples ◽

Polymerase Chain ◽

Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10-5 dilution of extracted tissue of artificially infected rhododendron ‘Cunningham's White’, and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

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Sky Segmentation for Enhanced Depth Reconstruction and Bokeh Rendering with Efficient Architectures

Electronic Imaging ◽

10.2352/issn.2470-1173.2020.14.coimg-378 ◽

2020 ◽

Vol 2020 (14) ◽

pp. 378-1-378-7

Author(s):

Tyler Nuanes ◽

Matt Elsey ◽

Radek Grzeszczuk ◽

John Paul Shen

Keyword(s):

Real Time ◽

Mobile Device ◽

Computational Cost ◽

False Positives ◽

Compact Model ◽

High Quality ◽

False Negatives ◽

Trade Off ◽

Depth Reconstruction ◽

Binary Classifiers

We present a high-quality sky segmentation model for depth refinement and investigate residual architecture performance to inform optimally shrinking the network. We describe a model that runs in near real-time on mobile device, present a new, highquality dataset, and detail a unique weighing to trade off false positives and false negatives in binary classifiers. We show how the optimizations improve bokeh rendering by correcting stereo depth misprediction in sky regions. We detail techniques used to preserve edges, reject false positives, and ensure generalization to the diversity of sky scenes. Finally, we present a compact model and compare performance of four popular residual architectures (ShuffleNet, MobileNetV2, Resnet-101, and Resnet-34-like) at constant computational cost.

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Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

Mansoura Veterinary Medical Journal ◽

10.35943/mvmj.2019.22.102 ◽

2019 ◽

Vol 20 (2) ◽

pp. 6-11

Author(s):

Aly El-Kenawy ◽

Mohamed El-Tholoth ◽

Emad A

Keyword(s):

Real Time ◽

Disease Virus ◽

Real Time Pcr ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Field Samples ◽

Feather Follicle ◽

Positive Results

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.

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