scholarly journals Detection and Diagnosis of Xylella fastidiosa by Specific Monoclonal Antibodies

Agronomy ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 48
Author(s):  
María Teresa Gorris ◽  
Antonio Sanz ◽  
Javier Peñalver ◽  
María M. López ◽  
Mario Colomer ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Real Time ◽  
Real Time Pcr ◽  
Polyclonal Antibodies ◽  
Xylella Fastidiosa ◽  
Low Cost ◽  
Kappa Index ◽  
Commercial Kits ◽  
O Antigens ◽  
Das Elisa

Monoclonal antibodies (MAb) specific to Xylella fastidiosa were obtained through hybridoma technology using heat-treated somatic O antigens from LMG 17159strain. Ten stable hybrydoma clones secreting MAb were selected and their isotype was determined. The MAbs 2G1/PPD, IgG1 showed specificity for X. fastidiosa, detecting all the analyzed strains representing different subspecies, STs and hosts. Polyclonal antibodies (PAb) against X. fastidiosa were also produced and antiserum 17159-O/IVIA was selected for the highest titre and its excellent detection capability. MAb 2G1/PPD was tested against strain IVIA 5235 in PBS and in spiked raw extract samples from almond, olive, citrus, and other hosts and its sensitivity by DAS-ELISA was 104 CFU mL−1. The MAb also reacted with high affinity and avidity against X. fastidiosa by DASI-ELISA and Tissue print-ELISA. The diagnostic parameters of DAS-ELISA based on MAb were calculated and compared with the gold standard real-time PCR. The diagnostic specificity of MAb2G1/PPD was 100%, the diagnostic sensitivity was 88.5% compared to Harper’s real-time PCR and 89.9% compared to Francis’ real-time PCR. The agreement between the techniques was almost perfect according to the estimated Cohen’s kappa-index, even in symptomless almond trees. The developed immunological techniques represent sustainable and low-cost analysis tools, based on specific, homogeneous, and well-characterized MAbs, which can be obtained in unlimited quantities in a reproducible way and constitute a guarantee for the standardization of commercial kits. They are a valuable option within a polyphasic strategy for the detection of X. fastidiosa.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

2015 ◽  
Author(s):  
Rainer Gransee ◽  
Tristan Schneider ◽  
Deniz Elyorgun ◽  
Xenia Strobach ◽  
Tobias Schunck ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Melting Curve ◽  
Lab On A Chip ◽  
Curve Analysis ◽  
Melting Curve Analysis

scholarly journals Real-Time Polymerase Chain Reaction for One-Hour On-Site Diagnosis of Pierce's Disease of Grape in Early Season Asymptomatic Vines

2002 ◽  
Vol 92 (7) ◽  
pp. 721-728 ◽  
Author(s):  
N. W. Schaad ◽  
D. Opgenorth ◽  
P. Gaush
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Xylella Fastidiosa ◽  
Molecular Techniques ◽  
Plant Diseases ◽  
Pierce's Disease ◽  
Chain Reaction ◽  
Pierce’S Disease ◽  
Polymerase Chain

Molecular-based techniques, such as polymerase chain reaction (PCR), can reduce the time needed for diagnosis of plant diseases when compared with classical isolation and pathogenicity tests. However, molecular techniques still require 2 to 3 days to complete. To the best of our knowledge, we describe for the first time a real-time PCR technique using a portable Smart Cycler for one-hour on-site diagnosis of an asymptomatic plant disease. Pierce's disease (PD) of grape, caused by the fastidious bacterium Xylella fastidiosa, causes serious losses in grapes in California and the southeastern United States. The disease has been difficult to diagnose because typical leaf scorching symptoms do not appear until late (June and after) in the season and the organism is very difficult to isolate early in the season. Sap and samples of macerated chips of secondary xylem from trunks of vines were used in a direct real-time PCR without extraction of DNA. Using two different sets of primers and probe, we diagnosed PD in 7 of 27 vines (26%) from four of six vineyards sampled 10 to 12 days after bud break in Kern, Tulare, and Napa counties of California. The diagnosis was confirmed by isolation of Xylella fastidiosa from two of the original PCR positive samples and later from symptomatic leaf petioles of four out of four vines from one vineyard that were originally PCR positive.

Low-Cost Monitoring of Campylobacter in Poultry Houses by Air Sampling and Quantitative PCR

2014 ◽  
Vol 77 (2) ◽  
pp. 325-330 ◽  
Author(s):  
M. S. R. SØNDERGAARD ◽  
M. H. JOSEFSEN ◽  
C. LÖFSTRÖM ◽  
L. S. CHRISTENSEN ◽  
K. WIECZOREK ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Air Sampling ◽  
Quantitative Risk Assessment ◽  
Quantitative Real Time Pcr ◽  
Airborne Particle ◽  
Air Samples ◽  
Particle Count ◽  
Poultry Houses

The present study describes the evaluation of a method for the quantification of Campylobacter by air sampling in poultry houses. Sampling was carried out in conventional chicken houses in Poland, in addition to a preliminary sampling in Denmark. Each measurement consisted of three air samples, two standard boot swab fecal samples, and one airborne particle count. Sampling was conducted over an 8-week period in three flocks, assessing the presence and levels of Campylobacter in boot swabs and air samples using quantitative real-time PCR. The detection limit for air sampling was approximately 100 Campylobacter cell equivalents (CCE)/m3. Airborne particle counts were used to analyze the size distribution of airborne particles (0.3 to 10 μm) in the chicken houses in relation to the level of airborne Campylobacter. No correlation was found. Using air sampling, Campylobacter was detected in the flocks right away, while boot swab samples were positive after 2 weeks. All samples collected were positive for Campylobacter from week 2 through the rest of the rearing period for both sampling techniques, although levels 1- to 2-log CCE higher were found with air sampling. At week 8, the levels were approximately 104 and 105 CCE per sample for boot swabs and air, respectively. In conclusion, using air samples combined with quantitative real-time PCR, Campylobacter contamination could be detected earlier than by boot swabs and was found to be a more convenient technique for monitoring and/or to obtain enumeration data useful for quantitative risk assessment of Campylobacter.

scholarly journals Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

2009 ◽  
Vol 107 (5) ◽  
pp. 1433-1439 ◽  
Author(s):  
K.L. Leung ◽  
C.W. Yip ◽  
W.F. Cheung ◽  
A.C.T. Lo ◽  
W.M. Ko ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Method

scholarly journals Confirmation of multi-parallel quantitative real-time PCR as the gold standard for detecting soil-transmitted helminths in stool

2021 ◽  
Author(s):  
Marina Papaiakovou ◽  
Nils Pilotte ◽  
Julia Dunn ◽  
David TJ Littlewood ◽  
Rubén O Cimino ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Low Cost ◽  
Strongyloides Stercoralis ◽  
Diagnostic Methods ◽  
Quantitative Real Time Pcr ◽  
Soil Transmitted Helminths ◽  
Stool Samples ◽  
Kappa Agreement

AbstractDue to its simplicity and cost-effectiveness, microscopy has seen extensive field-use as the diagnostic standard for the detection of soil-transmitted helminths (STH) in stool samples. However, the sensitivity of microscopy-based detection is inadequate in reduced-transmission settings where worm burden is oftentimes low. Equally problematic, eggs of closely related species oftentimes have indistinguishable morphologies, leading to species misidentification. In light of these shortcomings, the purpose of this study was to demonstrate multi-parallel quantitative real-time PCR (qPCR) as the new “gold standard” for STH detection. Accordingly, stool samples from non-endemic participants were spiked with limited numbers of eggs or larvae (1 to 40) of five different species of STH. DNA extracts were tested using two unique multi-parallel real-time PCR-based diagnostic methods. These methods employed different target sequences (ribosomal internal transcribed spacer, or highly repetitive non-coding regions), to evaluate the detection of DNA from as little as one egg per sample. There was a statistically significant kendall correlation between egg/larvae counts and qPCR from both methods for Trichuris trichiura (0.86 and 0.872 for NHM and Baylor assays) and a strong correlation (0.602 and 0.631 for NHM and Baylor assays, respectively) for Ascaris lumbricoides. Less strong but still significant was the Kendall Tau-b value for A. duodenale (0.408 for both) and for S. stercoralis (0.483 and 0.653, respectively). In addition, using field stool samples from rural Argentina both assays had fair to moderate kappa agreement (0.329-0.454), except for Strongyloides stercoralis (0.121) that both assays had slight agreement. In spite of the small cohort of samples, both qPCR assays, targeting of two independent genomic regions, provided reproducible results and we believe that, low cost multi-parallel quantitative real-time PCR-based diagnostics should supplant microscopy as the new gold standard for stool-based detection of soil transmitted helminths in public-health and community settings.

scholarly journals Toward point-of-care diagnostics of Candida auris

2020 ◽  
Author(s):  
Geoffrey Mulberry ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi ◽  
Brian N. Kim
Keyword(s):  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

scholarly journals Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR

2015 ◽  
Vol 142 (5) ◽  
pp. 555 ◽  
Author(s):  
BVishnu Bhat ◽  
DBenet Bosco Dhas ◽  
AHiasindh Ashmi ◽  
SubashChandra Parija ◽  
N Banupriya
Keyword(s):  
Real Time ◽  
Melting Temperature ◽  
Real Time Pcr ◽  
Low Cost ◽  
Snp Genotyping ◽  
Cycle Threshold ◽  
Allele Specific
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