scholarly journals shinyCurves, a shiny web application to analyse multisource qPCR amplification data: a COVID 19 case study

2020 ◽  
Author(s):  
S. Olaechea-Lázaro ◽  
I. García-Santisteban ◽  
JR. Pineda ◽  
I. Badiola ◽  
S. Alonso ◽  
...  
Keyword(s):  
Web Application ◽  
Automated System ◽  
Chain Reaction ◽  
Link Type ◽  
Qpcr Analysis ◽  
Standard Tool ◽  
Different Types ◽  
Polymerase Chain

AbstractSummaryQuantitative, reverse transcription polymerase chain reaction (qRT-PCR) has been the gold-standard tool for viral detection during the SARS-CoV-2 pandemic. However, the desperate rush for a quick diagnosis led the use of very different types of machines and proprietary software, leading to an unbearable complexity of data analysis with a limited parameter setup. Here, we present shinyCurves, a shiny web application created to analyse multisource qPCR amplification data from independent multi-plate format. Furthermore, our automated system allows the classification of the results as well as the plot of both amplification and melting curves. Altogether, our web application is an automated qPCR analysis resource available to the research community.AvailabilityThe shinyCurves web application to analyze multisource qPCR amplification data is publicly available under CC license (CC BY-NC-SA 4.0) at https://biosol.shinyapps.io/shinycurves/ and https://github.com/biosol/shinyCurves.

scholarly journals PCRedux: A Data Mining and Machine Learning Toolkit for qPCR Experiments

2021 ◽  
Author(s):  
Michał Burdukiewicz ◽  
Andrej-Nikolai Spiess ◽  
Dominik Rafacz ◽  
Konstantin Blagodatskikh ◽  
Jim Huggett ◽  
...  
Keyword(s):  
Machine Learning ◽  
Open Source Software ◽  
Working Environment ◽  
Learning And Development ◽  
Amplification Curve ◽  
Link Type ◽  
Qpcr Analysis ◽  
Precise Quantification

AbstractMotivationQuantitative Real-time PCR (qPCR) is a widely used -omics method for the precise quantification of nucleic acids, in which the result is associated with the presence/absence or quantity of a specific nucleic acid sequence. As the amount of qPCR data increases worldwide, the manual assessment of results becomes challenging and difficult to reproduce. To overcome this, some automatable characteristics of amplification curves have been described in the literature, often with an appropriate “rule of thumb”.ResultsWe developed PCRedux to analyze and calculate 90 numerical qPCR amplification curve descriptors (‘‘features”) from large datasets of qPCR amplification curves that are aimed for interpretable machine learning and development of decision support systems. In a case study of a diverse dataset with 3181 positive, negative and ambiguous amplification curves, as assessed by three human raters, we demonstrate a sensitivity >99 % and specificity >97 % in detecting positive and negative amplification. PCRedux is unique as it goes beyond traditional qPCR analysis to capture curvature properties that improve the characterization and classification of amplification curves. The calculation of the features is reproducible and objective, since R is used as a controllable working environment. PCRedux is not a black box, but open source software following on the principle of mathematically interpretable features. These can be combined with user-defined labels for automatic multi-category classification and regression in machine learning.Availabilityhttps://cran.r-project.org/package=PCRedux. Web server: http://shtest.evrogen.net/PCRedux-app/. Documentation: https://PCRuniversum.github.io/PCRedux/.

scholarly journals Molecular Characterization and Classification of Phytoplasmas Associated with Canola Yellows and a New Phytoplasma Strain Associated with Dandelions

Plant Disease ◽  
2001 ◽  
Vol 85 (1) ◽  
pp. 76-79 ◽  
Author(s):  
Keri Wang ◽  
Chuji Hiruki
Keyword(s):  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Universal Primers ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Aster Yellows ◽  
Distinct Subgroup ◽  
16S Ribosomal Dna ◽  
Polymerase Chain

DNA isolated from symptomatic canola (Brassica napus, Brassica rapa) and dandelion (Taraxacum officinale) was used to amplify 16S ribosomal DNA fragments by polymerase chain reaction using two pairs of universal primers P1/P6 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of the amplified DNA fragments using endonucleases AluI, HhaI, HpaII, MseI, RsaI, and Sau 3AI revealed two distinct types of phytoplasmas in canola with similar symptoms. One had the same RFLP profiles as the phytoplasmas in subgroup 16SrI-A, whereas the other one had RFLP profiles similar to those of phytoplasmas in subgroup 16SrI-B. Phytoplasmas were detected in symptomatic dandelion plants that were collected from canola and alfalfa fields where severe alfalfa witches'-broom occurred. Comparative studies indicated that two different phytoplasmas were associated with the dandelion plants. One was identified as a member of subgroup 16SrI-A, whereas another one was classified as a member of a distinct subgroup in the aster yellows group on the basis of the unique RFLP patterns.

Automated closed-vessel system for in vitro diagnostics based on polymerase chain reaction

1993 ◽  
Vol 39 (9) ◽  
pp. 1927-1933 ◽  
Author(s):  
J B Findlay ◽  
S M Atwood ◽  
L Bergmeyer ◽  
J Chemelli ◽  
K Christy ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Automated System ◽  
Closed Vessel ◽  
Chain Reaction ◽  
Panel Testing ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

Abstract An automated system for polymerase chain reaction (PCR) amplification and detection combats false-positive results caused by "PCR product carryover." The system uses a single vessel for both PCR amplification and the subsequent detection of PCR products, eliminating the need to handle PCR products in an open environment and risk product carryover. The sample and PCR reagents are introduced into one compartment within the vessel, and amplification occurs as they are thermally cycled. Other compartments contain the reagents for detection of PCR products. Pressure from a roller provides for sequential delivery of the contents of the compartments to a detection area. The PCR products are biotinylated at their 5' ends during amplification through the use of biotinylated primers. After delivery to the detection area, they are specifically captured by hybridization with immobilized oligonucleotide probes. Subsequent reaction with streptavidin-horseradish peroxidase conjugate forms a complex that catalyzes dye formation from dye precursor. Wash steps minimize nonspecific background. This format is amenable to multiplexing, permitting internal controls, speciation of bacteria, typing of viruses, and panel testing. An HIV assay performed with this system demonstrated 100% sensitivity and 95% specificity for 64 patients' samples relative to a conventional PCR assay based on 32P solution hybridization. Similarly, an automated closed-vessel assay of cytomegalovirus exhibited 97.5% sensitivity and 100% specificity.

Requirement Estimation and Design of Tag software in Web Application

2014 ◽  
Vol 9 (2) ◽  
pp. 1-19 ◽  
Author(s):  
Karan Gupta ◽  
Anita Goel
Keyword(s):  
Decision Process ◽  
Web Application ◽  
Design Document ◽  
Requirement Specification ◽  
Software Estimation

Tag software is included in a web application to facilitate categorization and classification of information. Generally, freely available tag software is adapted, or new code is written to incorporate tagging functionality. Since there is an absence of requirement specification and design document for tag software, even academically, it becomes difficult for the user to know about the possible features that can be included in the tag software. The user has to search for those features to be able to implement them in the software. So, there is a need that the user is made aware of the features available. Moreover, not all the features are relevant for the user; hence, there is a need for some kind of mechanism to ease the decision process. This paper presents - (1) a design for tag software, and (2) categorization of requirements of tag software in a web application. The design helps the developer during updating and analysis. The logical view of design displays interaction of entities and sub-entities with users. A weighted requirement checklist is presented which segregates features in three categories based on their popularity. This eases the task of selecting the requirement of tag software for the user. A metric, software estimation, is defined for quantifying selected requirements. A case study of freely available tag software is presented, in which estimation and design is applied.

scholarly journals Rapid simultaneous detection of multiple retroviral DNA sequences using the polymerase chain reaction and capillary DNA chromatography

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 879-886 ◽  
Author(s):  
FJ Sunzeri ◽  
TH Lee ◽  
RG Brownlee ◽  
MP Busch
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Blood Supply ◽  
Virus Type ◽  
Automated System ◽  
Type I ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Target Sequences ◽  
Nucleic Acid Sequences

Abstract The polymerase chain reaction (PCR) technique is a powerful new tool for amplifying target DNA, thus allowing for sensitive detection of specific nucleic acid sequences. One important potential use of PCR involves screening the donated blood supply for transfusion-transmitted viruses. Realization of this goal has been limited by (1) the requirement for multiple, discrete PCR reactions to amplify and detect target sequences of more than one virus, and (2) the lack of a rapid, nonhazardous means for specific detection of one or more PCR-amplified products. We report the simultaneous amplification of three distinct target sequences without discernable loss in sensitivity toward any single target sequence. We also demonstrate very rapid separation and detection of PCR-amplified viral DNA through the use of automated capillary DNA chromatography. Amplified DNA peaks were initially identified by scanning the capillary effluent at ultraviolet wavelengths, while discrimination of human immunodeficiency virus type 1 and human T-cell leukemic virus type I PCR-amplified DNA was accomplished through use of virus-specific, fluorescently labeled primers and probes. These results indicate progress toward an automated system for screening the blood supply for nucleic acid sequences of multiple pathogens.

scholarly journals Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones ofStaphylococcus aureus

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Salman Sahab Atshan ◽  
Mariana Nor Shamsudin ◽  
Zamberi Sekawi ◽  
Leslie Than Thian Lung ◽  
Rukman Awang Hamat ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Biofilm Formation ◽  
Clinical Information ◽  
Microtiter Plate ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Microtiter Plate Assay ◽  
High Prevalence ◽  
Different Types ◽  
Polymerase Chain

Clinical information about genotypically different clones of biofilm-producingStaphylococcus aureusis largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistantS. aureus(MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types ofspaand determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the samespatype were found to have similar properties in adheringto thepolystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes).icaADBCgenes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM andicaADBC) was confirmed by RT-PCR.

Classification of wheat PPO genes and effect of non-synonymous cSNP on kernel PPO activity

2008 ◽  
Vol 5 (1) ◽  
pp. 81-86 ◽  
Author(s):  
Wang Xiao-Bo ◽  
Ma Chuan-Xi ◽  
Si Hong-Qi ◽  
He Xian-Fang
Keyword(s):  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Chain Reaction ◽  
Wheat Varieties ◽  
Single Nucleotide ◽  
Pcr Product ◽  
Dnaman Software ◽  
Dna Fragment ◽  
Polymerase Chain

AbstractPolyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products. In this study, wheat PPO sequences (mRNA) were searched/BLASTed in the NCBI database and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into two clusters (I and II) and that three genes (‘i’) of cluster II seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNPs) were detected between two haplotypes of the PPO gene on chromosome 2D. Eighty of these were found in the coding region (coding (c) SNPs) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers (STS-H) were designed at some non-synonymous cSNPs sites and were used to investigate the correlations between allelic variants and PPO activity of seeds – a total of 130 common wheat varieties were evaluated in 2 years. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of a single dominance molecular marker, the multiplex polymerase chain reaction (PCR) system of STS-H and STS01 markers was also studied, based on the complementary between them.

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