scholarly journals Detection of Differential Host Susceptibility to the Marine Oomycete Pathogen Eurychasma dicksonii by Real-Time PCR: Not All Algae Are Equal

2008 ◽  
Vol 75 (2) ◽  
pp. 322-328 ◽  
Author(s):  
Claire M. M. Gachon ◽  
Martina Strittmatter ◽  
Dieter G. Müller ◽  
Julia Kleinteich ◽  
Frithjof C. Küpper
Keyword(s):  
Real Time ◽  
Brown Algae ◽  
Large Scale ◽  
Natural Populations ◽  
Host Susceptibility ◽  
Ecosystem Structure ◽  
Ectocarpus Siliculosus ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Oomycete Pathogen

ABSTRACT In the marine environment, a growing body of evidence points to parasites as key players in the control of population dynamics and overall ecosystem structure. However, their prevalence and impact on marine macroalgal communities remain virtually unknown. Indeed, infectious diseases of seaweeds are largely underdocumented, partly because of the expertise required to diagnose them with a microscope. Over the last few years, however, real-time quantitative PCR (qPCR) has emerged as a rapid and reliable alternative to visual symptom scoring for monitoring pathogens. Thus, we present here a qPCR assay suitable for the detection and quantification of the intracellular oomycete pathogen Eurychasma dicksonii in its ectocarpalean and laminarialean brown algal hosts. qPCR and microscopic observations made of laboratory-controlled cultures revealed that clonal brown algal strains exhibit different levels of resistance against Eurychasma, ranging from high susceptibility to complete absence of symptoms. This observation strongly argues for the existence of a genetic determinism for disease resistance in brown algae, which would have broad implications for the dynamics and genetic structure of natural populations. We also used qPCR for the rapid detection of Eurychasma in filamentous brown algae collected in Northern Europe and South America and found that the assay is specific, robust, and widely applicable to field samples. Hence, this study opens the perspective of combining large-scale disease monitoring in the field with laboratory-controlled experiments on the genome model seaweed Ectocarpus siliculosus to improve our understanding of brown algal diseases.

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

2010 ◽  
Vol 77 (1) ◽  
pp. 89-97 ◽  
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.

scholarly journals Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

Plant Disease ◽  
2020 ◽  
Vol 104 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Joseph D. Carrillo ◽  
Joey S. Mayorquin ◽  
Jason E. Stajich ◽  
Akif Eskalen
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Absolute Quantification ◽  
Coefficient Of Determination ◽  
Fungal Isolation ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Symbiotic Fungi ◽  
Fusarium Dieback ◽  
Platanus Racemosa

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

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

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.

Methods for the Quantification of Salivary Cortisol and of a-amylase in Biosensors and Portable Devices

2018 ◽  
Vol 68 (12) ◽  
pp. 2857-2859
Author(s):  
Cristina Mihaela Ghiciuc ◽  
Andreea Silvana Szalontay ◽  
Luminita Radulescu ◽  
Sebastian Cozma ◽  
Catalina Elena Lupusoru ◽  
...  
Keyword(s):  
Real Time ◽  
Medical Practice ◽  
Salivary Cortisol ◽  
Clinical Studies ◽  
Large Scale ◽  
Psychological Research ◽  
Portable Devices ◽  
Salivary Amylase ◽  
Specificity And Sensitivity

There is an increasing interest in the analysis of salivary biomarkers for medical practice. The objective of this article was to identify the specificity and sensitivity of quantification methods used in biosensors or portable devices for the determination of salivary cortisol and salivary a-amylase. There are no biosensors and portable devices for salivary amylase and cortisol that are used on a large scale in clinical studies. These devices would be useful in assessing more real-time psychological research in the future.

scholarly journals BattleNet: Capturing Advantageous Battlefield in RTS Games (Student Abstract)

2020 ◽  
Vol 34 (10) ◽  
pp. 13849-13850
Author(s):  
Donghyeon Lee ◽  
Man-Je Kim ◽  
Chang Wook Ahn
Keyword(s):  
Artificial Intelligence ◽  
Decision Making ◽  
Real Time ◽  
Large Scale ◽  
Outcome Predictor ◽  
Short Term ◽  
Rts Game

In a real-time strategy (RTS) game, StarCraft II, players need to know the consequences before making a decision in combat. We propose a combat outcome predictor which utilizes terrain information as well as squad information. For training the model, we generated a StarCraft II combat dataset by simulating diverse and large-scale combat situations. The overall accuracy of our model was 89.7%. Our predictor can be integrated into the artificial intelligence agent for RTS games as a short-term decision-making module.

scholarly journals Influence of extracorporeal membrane oxygenation on serum microRNA expression

2019 ◽  
Vol 47 (12) ◽  
pp. 6109-6119
Author(s):  
M. Scettri ◽  
H. Seeba ◽  
D. L. Staudacher ◽  
S. Robinson ◽  
D. Stallmann ◽  
...  
Keyword(s):  
Extracorporeal Membrane Oxygenation ◽  
Real Time ◽  
Underlying Disease ◽  
Real Time Quantitative Pcr ◽  
Membrane Oxygenation ◽  
Serum Microrna ◽  
Clinical Biomarkers ◽  
Differentially Expressed Mirnas ◽  
Before And After ◽  
Medical Intensive Care

Objective To date, no biomarkers have been established to predict haematological complications and outcomes of extracorporeal membrane oxygenation (ECMO). The aim of this study was to investigate the expression of a panel of microRNAs (miRNAs), which are promising biomarkers in many clinical fields, in patients before and after initiating ECMO. Methods Serum miRNA levels from 14 patients hospitalized for acute respiratory failure and supported with ECMO in our medical intensive care unit were analysed before and 24 hours after ECMO. In total, 179 serum-enriched miRNAs were profiled by using a real-time PCR panel. For validation, differentially expressed miRNAs were individually quantified with conventional real-time quantitative PCR at 0, 24, and 72 hours. Results Under ECMO support, platelet count significantly decreased by 65 × 103/µL (25th percentile = 154.3 × 103/µL; 75th percentile = 33 × 103/µL). Expression of the 179 miRNAs investigated in this study did not change significantly throughout the observational period. Conclusions According to our data, the expression of serum miRNAs was not altered by ECMO therapy itself. We conclude that ECMO does not limit the application of miRNAs as specific clinical biomarkers for the patients’ underlying disease.

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