scholarly journals New Molecular Tool for a Quick and Easy Detection of Apple Scab in the Field

Agronomy ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 581 ◽  
Author(s):  
Sara Franco Ortega ◽  
Simona Prencipe ◽  
Maria Lodovica Gullino ◽  
Davide Spadaro
Keyword(s):  
Direct Detection ◽  
Venturia Inaequalis ◽  
Apple Scab ◽  
Elongation Factor ◽  
Lamp Assay ◽  
Control Measures ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Sensitivity Testing ◽  
User Friendly

Venturia inaequalis, an agent of apple scab, is the most important pathogen of Malus x domestica. Control measures against this pathogen rely on intensive phytosanitary programs based on predictive models to identify the meteorological conditions conducive to the primary infection. The detection of the pathogen in field, both in naturally infected symptomatic and asymptomatic leaves, is desirable. Loop-mediated isothermal amplification (LAMP) assays are profitable molecular diagnostic tools for the direct detection of pathogens in field. A LAMP assay for V. inaequalis has been designed on the elongation factor 1-alpha sequence. The validation of the LAMP assay was carried out following the international EPPO standard PM 7/98 in terms of specificity, sensitivity, repeatability and reproducibility. Specificity testing was performed using target and non-target species, such as phylogenetically related Venturia species and other pathogens commonly found in apple, resulting in positive amplification only for the target with a time to positive ranging from 20 to 30 min. Sensitivity testing was performed with serial dilutions of DNA of the target and by artificial inoculation of young apple leaves. The reliability of the LAMP assay as an early-detection tool and its user-friendly application make it suitable for the diagnosis of apple scab in the field.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

scholarly journals Loop-mediated isothermal amplification (LAMP) : A rapid molecular diagnosis technique for detection of human pathogens

2017 ◽  
Vol 07 (03) ◽  
pp. 042-048
Author(s):  
Gunimala Chakraborty ◽  
Indrani Karunasagar ◽  
Anirban Chakraborty
Keyword(s):  
Treatment Strategies ◽  
Lamp Assay ◽  
Cost Effective ◽  
Isothermal Amplification ◽  
Current Status ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Pathogens ◽  
Quality Healthcare ◽  
Loop Mediated Isothermal Amplification

AbstractDelivery of quality healthcare in case of an infectious disease depends on how efficiently and how quickly the responsible pathogens are detected from the samples. Molecular methods can detect the presence of pathogens in a rapid and sensitive manner. Over the years, a number of such assays have been developed. However, these methods, although highly reliable and efficient, require use of expensive equipment, reagents, and trained personnel. Therefore, development of molecular assays that are simple, rapid, cost-effective, yet sensitive, is highly warranted to ensure efficient management or treatment strategies. Loop-mediated isothermal amplification (LAMP), a technique invented in the year 2000, is a novel method that amplifies DNA at isothermal conditions. Since its invention, this technique has been one of the most extensively used molecular diagnostic tools in the field of diagnostics offering rapid, accurate and cost-effective diagnosis of infectious diseases. Using the LAMP principle, many commercial kits have been developed in the last decade for a variety of human pathogens including bacteria, viruses and parasites. Currently LAMP assay is being considered as an effective diagnostic tool for use in developing countries because of its simple working protocol, allowing even an onsite application. The focus of this review is to describe the salient features of this technique the current status of development of LAMP assays with an emphasis on the pathogens of clinical significance.

scholarly journals Transcriptome Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa

2016 ◽  
Vol 60 (8) ◽  
pp. 4722-4733 ◽  
Author(s):  
Ariane Khaledi ◽  
Monika Schniederjans ◽  
Sarah Pohl ◽  
Roman Rainer ◽  
Ulrich Bodenhofer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Association Studies ◽  
Control Measures ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Learning Approaches ◽  
Content Type ◽  
Antibiotic Drug ◽  
The Future

ABSTRACTEmerging resistance to antimicrobials and the lack of new antibiotic drug candidates underscore the need for optimization of current diagnostics and therapies to diminish the evolution and spread of multidrug resistance. As the antibiotic resistance status of a bacterial pathogen is defined by its genome, resistance profiling by applying next-generation sequencing (NGS) technologies may in the future accomplish pathogen identification, prompt initiation of targeted individualized treatment, and the implementation of optimized infection control measures. In this study, qualitative RNA sequencing was used to identify key genetic determinants of antibiotic resistance in 135 clinicalPseudomonas aeruginosaisolates from diverse geographic and infection site origins. By applying transcriptome-wide association studies, adaptive variations associated with resistance to the antibiotic classes fluoroquinolones, aminoglycosides, and β-lactams were identified. Besides potential novel biomarkers with a direct correlation to resistance, global patterns of phenotype-associated gene expression and sequence variations were identified by predictive machine learning approaches. Our research serves to establish genotype-based molecular diagnostic tools for the identification of the current resistance profiles of bacterial pathogens and paves the way for faster diagnostics for more efficient, targeted treatment strategies to also mitigate the future potential for resistance evolution.

Electroflotation of Escherichia coli Improves Detection Rates by Loop-Mediated Isothermal Amplification

2018 ◽  
Vol 61 (4) ◽  
pp. 1209-1220
Author(s):  
Lena Michelle Diaz ◽  
Daniel Jenkins ◽  
Ryo Kubota ◽  
Natalie Walter ◽  
Yong Li ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Pathogen Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
Lamp Assay ◽  
Molecular Diagnostic ◽  
Process Conditions ◽  
Diagnostic Systems ◽  
Detection Rates ◽  
Glycerate Kinase

Abstract. The power of portable molecular diagnostic systems for detection of pathogenic microorganisms in food and environmental samples is largely limited by small assay volumes (typically 1 to 5 µL), making direct detection of trace contamination (i.e., <104 CFU mL-1) unreliable. To improve detection limits for pathogens dispersed on an ecological scale, we developed a portable point-of-care (POC) sample preparation system using electroflotation (EF) to recover small quantities of these organisms from samples of hundreds of milliliters. Electrolysis reactions, supported on platinum-coated titanium electrodes, generate hydrogen and oxygen microbubbles that impel and displace suspended cells into a recovered concentrate. Samples were prepared by inoculating 380 mL of sterilized phosphate buffer (0.1 M, pH 6.6) with stock culture of ATCC 25922 to final concentrations ranging from 102 to 104 CFU mL-1. Samples were subjected to 10, 15, and 20 min durations of EF treatment under high and low turbulence conditions. We used a loop-mediated amplification (LAMP) assay with primers targeting a single-copy gene (glycerate kinase) in generic to evaluate the effects of EF treatment on concentration and recovery of detectable cell material. LAMP failed to detect in all untreated (control) samples at concentrations below 104 CFU mL-1 but was able to detect in 102 CFU mL-1 samples subjected to various conditions of EF treatment. Two-way ANOVA showed significant differences in detection rates between EF treatment durations for both high (p = 0.0019) and low turbulence (p = 0.002). Dunnett’s multiple comparison tests identified five process conditions resulting in significant (p < 0.05) differences in detection between treatments and the control. Keywords: Biotechnology, Electrolysis, Food pathogens, Microbubbles, Molecular diagnostics, Pathogen detection, POC sample preparation.

scholarly journals Genetic Diversity and Gene Flow of Four South African Venturia inaequalis (Apple Scab) Populations

2017 ◽  
Vol 107 (4) ◽  
pp. 455-462 ◽  
Author(s):  
T. A. Koopman ◽  
J. C. Meitz-Hopkins ◽  
A. E. Bester-van der Merwe ◽  
K. R. Tobutt ◽  
C. Bester ◽  
...  
Keyword(s):  
South Africa ◽  
Gene Flow ◽  
Ssr Markers ◽  
Gene Diversity ◽  
Venturia Inaequalis ◽  
Large Subunit ◽  
Apple Scab ◽  
Elongation Factor ◽  
Climatic Conditions ◽  
Rpb1 Gene

Venturia inaequalis isolates were collected during the 2012/13 and 2013/14 seasons from the four principal apple growing regions of South Africa, Elgin (n = 114), Koue Bokkeveld (n = 126), Lower Langkloof (n = 92), and Upper Langkloof (n = 103). Sequence analysis of the ribosomal internal transcribed spacer (ITS) gene regions and genotyping with six (2012/13) and seven (2013/14) microsatellite (SSR) markers was conducted. A subset of 12 isolates from the individual ITS haplotype groups were sequenced for the translation elongation factor-1 alpha (TEF1) and the large subunit of the RNA polymerases II (RPB1) gene regions. Four haplotypes were found for ITS, whereas all isolates were identical for the TEF1 and RPB1 gene regions. The SSR markers revealed considerable variation with an average gene diversity (H) of 0.675. Multivariate analysis (discriminant analysis of principal components [DAPC]) revealed that the two Langkloof populations clustered together with the Koue Bokkeveld population. The population from the warmer winter region, Elgin, clustered separately from the rest of the populations (ΦPT = 0.076 to 0.116; P ≤ 0.05). Estimates of gene flow showed the highest migration rate from the Koue Bokkeveld, toward the Lower Langkloof (M = 151.1), and the least migration to and from the Elgin region (average M = 42.75). Occasionally, identical genotypes (clones) were detected across seasons in the Koue Bokkeveld and Elgin area, which might contribute to overwintering conidia. From this study, it is evident that South Africa most likely has V. inaequalis subpopulations linked to diverse climatic conditions of the coastal Elgin region compared with the mountainous inland regions of the Koue Bokkeveld and the Langkloof.

scholarly journals Genome resources of two pathotypes of the potato cyst nematode Globodera rostochiensis from New York

2020 ◽  
Author(s):  
Xiaohong Wang ◽  
Huijun Yang ◽  
Pierre-Yves Veronneau ◽  
David Thurston ◽  
Benjamin Mimee
Keyword(s):  
New York ◽  
New York State ◽  
Potato Production ◽  
Globodera Rostochiensis ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Control Measures ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Original Field

The potato cyst nematode, Globodera rostochiensis, is a regulated pest posing a serious threat to potato production worldwide. Although the endemic pathotype (Ro1) of G. rostochiensis has been confined to New York State for several decades as a result of quarantine regulations and management with resistant potato cultivars, a virulent pathotype, Ro2, has emerged, for which control measures are scarce. The ability to detect Ro2 early in fields is necessary to sustain the success of G. rostochiensis quarantine in the U.S. Here, we report the comparative analysis of whole genome sequences of multiple single-cyst-derived Ro1 and Ro2 lines, propagated from original field populations. The identified discriminant variants are good targets for developing molecular diagnostic tools for differentiating G. rostochiensis pathotypes in NY.

scholarly journals Multilocus Sequence Analysis of Selected Housekeeping- and Pathogenicity-Related Genes in Venturia inaequalis

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 447
Author(s):  
Monika Michalecka ◽  
Joanna Puławska
Keyword(s):  
Resistance Genes ◽  
Venturia Inaequalis ◽  
Apple Scab ◽  
Elongation Factor ◽  
Housekeeping Genes ◽  
Scab Resistance ◽  
Phylogenetic Position ◽  
Phylogenetic Groups ◽  
Evolutionary Forces ◽  
Genes Encoding

The relationship between housekeeping and pathogenicity-related genes and virulence or avirulence towards the primary Malus resistance genes (R) has not been previously studied for Venturia inaequalis fungus, the causal agent of apple scab. In this study, the sequences of two housekeeping genes encoding elongation factor alpha (EF-1α) and β-tubulin and two previously unstudied effector genes of V. inaequalis from mannosidase and glucosidase families of 100 strains collected from apple cultivars with Rvi6, Rvi1, and Rvi17 and without known scab resistance genes were submitted to the analyses. Based on the phylogenetic and diversity data, as well as recombination analyses of the sequenced regions, we assessed the phylogenetic relationships and genetic structure of the pathogen within the species and the evolutionary forces that are currently acting upon this microorganism. The topology of the obtained phylograms demonstrates the lack of a relationship between the phylogenetic position of the strain and the host cultivar and the geographical origin or race of the strain. The isolates from different hosts were differentiated but did not form diagnosable, distinct phylogenetic groups. These results suggest that the analyzed genes may be too conserved to reflect the adaptation of pathogens to apple genotypes with different R genes; thus, they do not adequately reflect race discrimination. In contrast, based on variation and gene flow estimation, genetic divergence was observed among strains virulent to apple trees containing Rvi6. The results of this study confirmed a lack of free recombination between strains and demonstrated that the analyzed regions are in linkage disequilibrium and contain non-random polymorphisms associated with the strain.

Constituents from Morus root bark against Venturia inaequalis – the causal agent of apple scab

Planta Medica ◽  
2007 ◽  
Vol 73 (09) ◽  
Author(s):  
JM Rollinger ◽  
R Spitaler ◽  
M Menz ◽  
P Schneider ◽  
EP Ellmerer ◽  
...  
Keyword(s):  
Venturia Inaequalis ◽  
Apple Scab ◽  
Causal Agent ◽  
Root Bark

scholarly journals 630. A 5-mRNA host response whole-blood classifier trained using patients with non-COVID-19 viral infections accurately predicts severity of COVID-19

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S375-S376
Author(s):  
ljubomir Buturovic ◽  
Purvesh Khatri ◽  
Benjamin Tang ◽  
Kevin Lai ◽  
Win Sen Kuan ◽  
...  
Keyword(s):  
Logistic Regression ◽  
Respiratory Failure ◽  
Viral Infections ◽  
Lamp Assay ◽  
Training Data ◽  
Diagnostic Tools ◽  
Severe Respiratory Failure ◽  
Proof Of Concept ◽  
Risk Of Death ◽  
Qpcr Platform

Abstract Background While major progress has been made to establish diagnostic tools for the diagnosis of SARS-CoV-2 infection, determining the severity of COVID-19 remains an unmet medical need. With limited hospital resources, gauging severity would allow for some patients to safely recover in home quarantine while ensuring sicker patients get needed care. We discovered a 5 host mRNA-based classifier for the severity of influenza and other acute viral infections and validated the classifier in COVID-19 patients from Greece. Methods We used training data (N=705) from 21 retrospective clinical studies of influenza and other viral illnesses. Five host mRNAs from a preselected panel were applied to train a logistic regression classifier for predicting 30-day mortality in influenza and other viral illnesses. We then applied this classifier, with fixed weights, to an independent cohort of subjects with confirmed COVID-19 from Athens, Greece (N=71) using NanoString nCounter. Finally, we developed a proof-of-concept rapid, isothermal qRT-LAMP assay for the 5-mRNA host signature using the QuantStudio 6 qPCR platform. Results In 71 patients with COVID-19, the 5 mRNA classifier had an AUROC of 0.88 (95% CI 0.80-0.97) for identifying patients with severe respiratory failure and/or 30-day mortality (Figure 1). Applying a preset cutoff based on training data, the 5-mRNA classifier had 100% sensitivity and 46% specificity for identifying mortality, and 88% sensitivity and 68% specificity for identifying severe respiratory failure. Finally, our proof-of-concept qRT-LAMP assay showed high correlation with the reference NanoString 5-mRNA classifier (r=0.95). Figure 1. Validation of the 5-mRNA classifier in the COVID-19 cohort. (A) Expression of the 5 genes used in the logistic regression model in patients with (red) and without (blue) mortality. (B) The 5-mRNA classifier accurately distinguishes non-severe and severe patients with COVID-19 as well as those at risk of death. Conclusion Our 5-mRNA classifier demonstrated very high accuracy for the prediction of COVID-19 severity and could assist in the rapid, point-of-impact assessment of patients with confirmed COVID-19 to determine level of care thereby improving patient management and healthcare burden. Disclosures ljubomir Buturovic, PhD, Inflammatix Inc. (Employee, Shareholder) Purvesh Khatri, PhD, Inflammatix Inc. (Shareholder) Oliver Liesenfeld, MD, Inflammatix Inc. (Employee, Shareholder) James Wacker, n/a, Inflammatix Inc. (Employee, Shareholder) Uros Midic, PhD, Inflammatix Inc. (Employee, Shareholder) Roland Luethy, PhD, Inflammatix Inc. (Employee, Shareholder) David C. Rawling, PhD, Inflammatix Inc. (Employee, Shareholder) Timothy Sweeney, MD, Inflammatix, Inc. (Employee)

scholarly journals Can Traditional Chinese Medicine Diagnosis Be Parameterized and Standardized? A Narrative Review

Healthcare ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 177
Author(s):  
Luís Carlos Matos ◽  
Jorge Pereira Machado ◽  
Fernando Jorge Monteiro ◽  
Henry Johannes Greten
Keyword(s):  
Chinese Medicine ◽  
Traditional Chinese Medicine ◽  
Functional Status ◽  
Control Measures ◽  
Systematic Evaluation ◽  
Diagnostic Tools ◽  
Functional Evaluation ◽  
Diagnostic Systems ◽  
Mechanical Pulse ◽  
Proof Of Efficacy

The integration of Traditional Chinese Medicine (TCM) in Western health systems and research requires a rational communicable theory, scientific proof of efficacy and safety, and quality control measures. The existence of clear definitions and the diagnosis standardization are critical factors to establish the patient’s vegetative functional status accurately and, therefore, systematically apply TCM therapeutics such as the stimulation of reflex skin areas known as acupoints. This science-based conceptualization entails using validated methods, or even developing new systems able to parameterize the diagnosis and assess TCM related effects by objective measurements. Traditionally, tongue and pulse diagnosis and the functional evaluation of action points by pressure sensitivity and physical examination may be regarded as essential diagnostic tools. Parameterizing these techniques is a future key point in the objectification of TCM diagnosis, such as by electronic digital image analysis, mechanical pulse diagnostic systems, or the systematic evaluation of acupoints’ electrophysiology. This review aims to demonstrate and critically analyze some achievements and limitations in the clinical application of device-assisted TCM diagnosis systems to evaluate functional physiological patterns. Despite some limitations, tongue, pulse, and electrophysiological diagnosis devices have been reported as a useful tool while establishing a person’s functional status.

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