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Methods in Molecular Biology (Clifton,... 2022Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for...
Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for viroid diagnosis. In the amplification mixtures, several pairs of primers work together in the same conditions to detect different targets. Here, we describe the development and use of a multiplex reverse transcription polymerase chain reaction protocol highlighting the most crucial factors that can significantly affect the quality of the method. First, particular attention must be paid to primer design. Then, the amplification mixture and temperature conditions must be calibrated precisely to avoid cross reactivity or loss in sensitivity. Finally, the detection system of the amplification results must allow a specific identification of the amplified target(s).
Topics: Multiplex Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Viroids
PubMed: 34845694
DOI: 10.1007/978-1-0716-1464-8_15 -
Cold Spring Harbor Protocols May 2018This protocol describes the reagents and procedures required to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase....
This protocol describes the reagents and procedures required to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase. It is the foundation for all subsequent variations of the polymerase chain reaction (PCR).
Topics: DNA; Nucleic Acid Denaturation; Polymerase Chain Reaction
PubMed: 29717051
DOI: 10.1101/pdb.prot095117 -
Trends in Biotechnology Jul 2019Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the... (Review)
Review
Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
Topics: Costs and Cost Analysis; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Time
PubMed: 30654913
DOI: 10.1016/j.tibtech.2018.12.002 -
Biosensors May 2021Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR)... (Review)
Review
Molecular diagnostics has been the front runner in the world's response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT-PCR POC systems approved by various agencies for COVID-19 detection are discussed.
Topics: Animals; COVID-19; COVID-19 Nucleic Acid Testing; CRISPR-Cas Systems; Equipment Design; Humans; Point-of-Care Testing; Polymerase Chain Reaction; SARS-CoV-2
PubMed: 34062874
DOI: 10.3390/bios11050141 -
Methods in Molecular Biology (Clifton,... 2017Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the... (Review)
Review
Quantitative polymerase chain reaction (qPCR) is a routinely used method for the detection and quantitation of gene expression in real time. Multiplex qPCR requires the use of probe-based assays, in which each probe is labeled with a unique fluorescent dye, resulting in different observed colors for each assay. The signal from each dye is used to quantitate the amount of each target separately in the same tube or well. The availability to multiplex therefore allows the measurement of the expression levels of several targets or genes of interest quickly. Here, we describe a method using the SensiFAST and SensiFAST One-Step probe kits which allows simultaneous real-time quantitation of up to 5 amplicons.
Topics: Biomarkers; Humans; Molecular Diagnostic Techniques; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity
PubMed: 27896761
DOI: 10.1007/978-1-4939-6730-8_8 -
Mini Reviews in Medicinal Chemistry 2023Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the... (Review)
Review
Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.
Topics: Humans; Molecular Biology; Water; Freeze Drying; Polymerase Chain Reaction; Cryopreservation; Point-of-Care Systems
PubMed: 35927908
DOI: 10.2174/1389557522666220802144057 -
Cold Spring Harbor Protocols Oct 2018In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is...
In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template. The ability to quantify the amplified DNA during the exponential phase of the PCR, when none of the components of the reaction is limiting, has resulted in dramatically improved precision in the quantitation of target sequences. In addition, because of the high sensitivity of fluorometric detection, real-time PCR is capable of measuring the initial concentration of target DNA over a vast dynamic range (up to eight or nine orders of magnitude) and with a high degree of sensitivity (as little as one copy of template DNA). Although it is a powerful technique, researchers often face challenges in reliability and reproducibility because of the lack of assay standardization. Therefore, it is critical to optimize the reagents and reaction conditions, include proper internal and external controls, and perform rigorous data analysis in order to generate accurate and reproducible results in real-time PCR experiments.
Topics: DNA; Guidelines as Topic; Real-Time Polymerase Chain Reaction; Reference Standards
PubMed: 30275081
DOI: 10.1101/pdb.top095000 -
Clinical Chemistry Aug 2020Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013,...
Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.
Topics: Guidelines as Topic; Humans; Nucleic Acids; Polymerase Chain Reaction; Publications
PubMed: 32746458
DOI: 10.1093/clinchem/hvaa125 -
Methods in Molecular Biology (Clifton,... 2016Emulsion PCR (EmPCR) is a commonly employed method for template amplification in multiple NGS-based sequencing platforms. The basic principle of emPCR is dilution and...
Emulsion PCR (EmPCR) is a commonly employed method for template amplification in multiple NGS-based sequencing platforms. The basic principle of emPCR is dilution and compartmentalization of template molecules in water droplets in a water-in-oil emulsion. Ideally, the dilution is to a degree where each droplet contains a single template molecule and functions as a micro-PCR reactor. Here, we discuss the basic principles, advantages, and challenges of applications of emPCR in clinical testing. We describe the methods of preparation and enrichment of template-positive Ion PGM™ Template OT2 200 Ion Sphere™ Particles (ISPs) on the Ion Personal Genome Machine(®) (PGM™) System. For routine clinical testing, following library generation, we employ the automated Ion OneTouch™ System that includes the Ion OneTouch™ 2 and the Ion OneTouch™ ES instruments for template generation and enrichment of template-positive ISPs, respectively.
Topics: Emulsions; Humans; Polymerase Chain Reaction
PubMed: 26843044
DOI: 10.1007/978-1-4939-3360-0_4 -
Cold Spring Harbor Protocols Oct 2018This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR...
This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR volume of 20 µL (although most manufacturers recommend 50-µL reactions). However, if the PCR target is not very abundant (i.e., present at one to 10 copies per sample), a larger volume may yield better reproducibility between samples. Discussion on preparing high-quality RNA, choosing a priming method, selecting an enzyme, and selecting an endogenous reference gene is also included.
Topics: RNA; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription
PubMed: 30275077
DOI: 10.1101/pdb.prot095042