scholarly journals Development of high-resolution melting PCR (HRM-PCR) assay to identify native fungal species associated with the wheat endosphere

2020 ◽  
Vol 61 (4) ◽  
pp. 629-635
Author(s):  
Tomasz Cłapa ◽  
Katarzyna Mikołajczak ◽  
Lidia Błaszczyk ◽  
Dorota Narożna
Keyword(s):  
High Resolution ◽  
Large Scale ◽  
High Resolution Melting ◽  
Low Cost ◽  
Fungal Species ◽  
Molecular Techniques ◽  
Pcr Assay ◽  
Fungal Isolates ◽  
Large Scale Screening

Abstract Understanding the complexity and biodiversity of fungal communities associated with the wheat endosphere can facilitate the identification of novel strains that might be beneficial to the host plant. However, the differentiation and taxonomic classification of the endosphere-associated fungi with respect to various cultivars and plant organs are challenging, time-consuming, and expensive, even with the use of molecular techniques. In the present work, we describe a fast, simple, and low-cost method based on high-resolution melting PCR (HRM-PCR) for the identification and differentiation of wheat endogenous fungal isolates. Using this approach, we differentiated 28 fungal isolates, which belonged to five different genera, namely Alternaria, Penicillium, Epicoccum, Fusarium, and Trichoderma. Furthermore, the results of the study revealed that this method can allow large-scale screening of cultured samples.

scholarly journals Analytical Validation and Clinical Application of Rapid Serological Tests for SARS-CoV-2 Suitable for Large-Scale Screening

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 869
Author(s):  
Amedeo De Nicolò ◽  
Valeria Avataneo ◽  
Jessica Cusato ◽  
Alice Palermiti ◽  
Jacopo Mula ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Real Time Pcr ◽  
Large Scale ◽  
High Sensitivity ◽  
Analytical Validation ◽  
Pcr Assay ◽  
Serological Tests ◽  
Flow Test ◽  
Lateral Flow Test ◽  
Large Scale Screening

Recently, large-scale screening for COVID-19 has presented a major challenge, limiting timely countermeasures. Therefore, the application of suitable rapid serological tests could provide useful information, however, little evidence regarding their robustness is currently available. In this work, we evaluated and compared the analytical performance of a rapid lateral-flow test (LFA) and a fast semiquantitative fluorescent immunoassay (FIA) for anti-nucleocapsid (anti-NC) antibodies, with the reverse transcriptase real-time PCR assay as the reference. In 222 patients, LFA showed poor sensitivity (55.9%) within two weeks from PCR, while later testing was more reliable (sensitivity of 85.7% and specificity of 93.1%). Moreover, in a subset of 100 patients, FIA showed high sensitivity (89.1%) and specificity (94.1%) after two weeks from PCR. The coupled application for the screening of 183 patients showed satisfactory concordance (K = 0.858). In conclusion, rapid serological tests were largely not useful for early diagnosis, but they showed good performance in later stages of infection. These could be useful for back-tracing and/or to identify potentially immune subjects.

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe

2021 ◽  
Vol 90 ◽  
pp. 104741
Author(s):  
Maëllys Kevin ◽  
Guillaume Girault ◽  
Yvan Caspar ◽  
Moulay Ali Cherfa ◽  
Christiane Mendy ◽  
...  
Keyword(s):  
High Resolution ◽  
Western Europe ◽  
High Resolution Melting ◽  
Epidemiological Surveillance ◽  
Pcr Assay

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.

scholarly journals High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study

2015 ◽  
Vol 4 (2) ◽  
pp. 73-81 ◽  
Author(s):  
Marco Marino ◽  
Maria Laura Monzani ◽  
Giulia Brigante ◽  
Katia Cioni ◽  
Bruno Madeo ◽  
...  
Keyword(s):  
Cohort Study ◽  
High Resolution ◽  
Molecular Analysis ◽  
Prospective Cohort Study ◽  
Prospective Cohort ◽  
Large Scale ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Braf V600e Mutation ◽  
Braf V600e

Objective: The diagnostic accuracy of thyroid fine needle aspiration biopsy (FNAB) can be improved by the combination of cytological and molecular analysis. In this study, washing liquids of FNAB (wFNAB) were tested for the BRAF V600E mutation, using the sensitive and cost-effective technique called high-resolution melting (HRM). The aim was to demonstrate the feasibility of BRAF analysis in wFNAB and its diagnostic utility, combined with cytology. Design: Prospective cohort study. Methods: 481 patients, corresponding to 648 FNAB samples, were subjected to both cytological (on cells smeared onto a glass slide) and molecular analysis (on fluids obtained washing the FNAB needle with 1 ml of saline) of the same aspiration. BRAF V600E analysis was performed by HRM after methodological validation for application to wFNAB (technique sensitivity: 5.4%). Results: The cytological results of the FNAB were: 136 (21%) nondiagnostic (THY1); 415 (64%) benign (THY2); 80 (12.4%) indeterminate (THY3); 9 (1.4%) suspicious for malignancy (THY4); 8 (1.2%) diagnostic of malignancy (THY5). The BRAF V600E mutation was found in 5 THY2, 2 THY3, 6 THY4 and 6 THY5 samples. Papillary carcinoma diagnosis was histologically confirmed in all BRAF+ thyroidectomized patients. BRAF combined with cytology improved the diagnostic value compared to cytology alone in a subgroup of 74 operated patients. Conclusions: HRM was demonstrated to be a feasible method for BRAF analysis in wFNAB. Thanks to its sensitivity and cost-effectiveness, it might be routinely used on a large scale in clinical practice. In perspective, standby wFNAB samples could be analyzed a posteriori in case of indeterminate cytology and/or suspicious findings on ultrasound.

scholarly journals Development and application of affordable SNP typing approaches to genotype Mycobacterium tuberculosis complex strains in low and high burden countries

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Irving Cancino-Muñoz ◽  
Ana Gil-Brusola ◽  
Manuela Torres-Puente ◽  
Carla Mariner-Llicer ◽  
John Dogba ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Low Income ◽  
Sanger Sequencing ◽  
High Resolution Melting ◽  
Mycobacterium Tuberculosis Complex ◽  
Molecular Techniques ◽  
Low Income Countries ◽  
Tuberculosis Complex ◽  
Typing Methods

Abstract The Mycobacterium tuberculosis complex (MTBC) comprises the species that causes tuberculosis (TB) which affects 10 million people every year. A robust classification of species, lineages, and sub-lineages is important to explore associations with drug resistance, epidemiological patterns or clinical outcomes. We present a rapid and easy-to-follow methodology to classify clinical TB samples into the main MTBC clades. Approaches are based on the identification of lineage and sub-lineage diagnostic SNP using a real-time PCR high resolution melting assay and classic Sanger sequencing from low-concentrated, low quality DNA. Thus, suitable for implementation in middle and low-income countries. Once we validated our molecular procedures, we characterized a total of 491 biological samples from human and cattle hosts, representing countries with different TB burden. Overall, we managed to genotype ~95% of all samples despite coming from unpurified and low-concentrated DNA. Our approach also allowed us to detect zoonotic cases in eight human samples from Nigeria. To conclude, the molecular techniques we have developed, are accurate, discriminative and reproducible. Furthermore, it costs less than other classic typing methods, resulting in an affordable alternative method in TB laboratories.

Temperature evolution of an experimental salt-gradient solar pond

2010 ◽  
Vol 1 (4) ◽  
pp. 246-250 ◽  
Author(s):  
F. Suárez ◽  
A. E. Childress ◽  
S. W. Tyler
Keyword(s):  
High Resolution ◽  
Large Scale ◽  
Thermal Evolution ◽  
Low Cost ◽  
Heat Extraction ◽  
Low Grade ◽  
Distributed Temperature Sensing ◽  
Solar Pond ◽  
Thermal Desalination ◽  
Salt Gradient

A salt-gradient solar pond is a low-cost, large-scale solar collector with integrated storage that can be used as a source of energy in low-grade-heat thermal desalination systems. This work presents the thermal evolution of an experimental solar pond for both the maturation and heat extraction time periods. The temperature profile was measured every 1.1 cm using a vertical high-resolution distributed temperature sensing (DTS) system, with a temperature resolution of 0.04ºC. Temperatures of 34 and 45ºC were achieved in the bottom of the pond when the lights were on for 12 and 24 hours per day, respectively. Heat was extracted at a rate of 139 W from the solar pond, which corresponded to an efficiency of 29%. Stratification and mixing were clearly observed inside the solar pond using the vertical high-resolution DTS system.

scholarly journals Rapid, Reliable and Robust approach for extraction-free RT-PCR based detection of SARS-CoV-2 in clinical setting to expedite large scale screening

2021 ◽  
Author(s):  
Abhilasha Dubey ◽  
Sanjay Upadhyay ◽  
Manjeet Mehta
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
Low Cost ◽  
Diagnostic Methods ◽  
Analysis Method ◽  
Rt Pcr ◽  
Qpcr Method ◽  
Quantitative Results ◽  
Pooled Samples ◽  
Large Scale Screening

Rapid, reliable and robust method for the detection of SARS-CoV-2 is an indispensable need for diagnostics. The development of diagnostic methods will aid to address further waves of the pandemic potentially with rapid surveillance of disease and to allay the fears. To meet this challenge, we have developed a rapid RT-qPCR method for the detection of 3 target genes or confirmatory genes in less than 30 minutes. The assay showed 100% sensitivity and 100% specificity when tested on 120 samples. We compared a conventional extraction based method with extraction-free method, and then further reduced the run time of extraction free method. Additionally, we have validated our rapid RT-qPCR method for the assessment of pooled samples. We hereby propose a most reliable approach for the mass screening of samples with ease of operation at a low cost. Finally we designed a single tube analysis method which provides qualitative as well as quantitative results in minimum time.

scholarly journals Massive Multiplexing Can Deliver a $1 Test for COVID-19

2020 ◽  
Author(s):  
Paul DN Hebert ◽  
Sean WJ Prosser ◽  
Natalia V Ivanova ◽  
Evgeny V Zakharov ◽  
Sujeevan Ratnasingham
Keyword(s):  
Large Scale ◽  
Low Cost ◽  
Rna Extraction ◽  
Respiratory Syndrome Virus ◽  
Major Advance ◽  
Rt Pcr ◽  
Screening Programs ◽  
Global Pandemic ◽  
Gene Coding ◽  
Large Scale Screening

ABSTRACTThe severe acute respiratory syndrome virus, SARS-CoV-2 (hereafter COVID-19), rapidly achieved global pandemic status, provoking large-scale screening programs in many nations. Their activation makes it imperative to identify methods that can deliver a diagnostic result at low cost. This paper describes an approach which employs sequence variation in the gene coding for its envelope protein as the basis for a scalable, inexpensive test for COVID-19. It achieves this by coupling a simple RNA extraction protocol with low-volume RT-PCR, followed by E-Gel screening and sequencing on high-throughput platforms to analyze 10,000 samples in a run. Slight modifications to the protocol could support screening programs for other known viruses and for viral discovery. Just as the $1,000 genome is transforming medicine, a $1 diagnostic test for viral and bacterial pathogens would represent a major advance for public health.

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