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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 -
Bioscience Reports Dec 2018Polymerase chain reaction (PCR) is a molecular biology technique used to multiply certain deoxyribonucleic acid (DNA) fragments. It is a common and indispensable... (Review)
Review
Polymerase chain reaction (PCR) is a molecular biology technique used to multiply certain deoxyribonucleic acid (DNA) fragments. It is a common and indispensable technique that has been applied in many areas, especially in clinical laboratories. The third generation of polymerase chain reaction, droplet digital polymerase chain reaction (ddPCR), is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify DNA. Droplet digital polymerase chain reaction is now widely used in low-abundance nucleic acid detection and is useful in diagnosis of infectious diseases. Here, we summarized the potential advantages of droplet digital polymerase chain reaction in clinical diagnosis of infectious diseases, including viral diseases, bacterial diseases and parasite infections, concluded that ddPCR provides a more sensitive, accurate, and reproducible detection of low-abundance pathogens and may be a better choice than quantitative polymerase chain reaction for clinical applications in the future.
Topics: Communicable Diseases; DNA; Humans; Molecular Diagnostic Techniques; Polymerase Chain Reaction
PubMed: 30341241
DOI: 10.1042/BSR20181170 -
Journal of Clinical Microbiology Jun 2017Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute... (Review)
Review
Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This minireview will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology, and considerations for implementation of the method in a clinical laboratory.
Topics: Communicable Diseases; Humans; Molecular Diagnostic Techniques; Polymerase Chain Reaction
PubMed: 28298452
DOI: 10.1128/JCM.00211-17 -
Human Gene Therapy Aug 2023Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of...
Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID) assay is an cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.
Topics: Humans; Dependovirus; Polymerase Chain Reaction; Genome, Viral; Real-Time Polymerase Chain Reaction
PubMed: 37276150
DOI: 10.1089/hum.2023.014 -
Emerging Microbes & Infections Dec 2021SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens,...
SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; COVID-19; COVID-19 Testing; Coinfection; Female; Humans; Influenza, Human; Male; Middle Aged; Multiplex Polymerase Chain Reaction; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Respiratory Syncytial Virus Infections; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Young Adult
PubMed: 33410371
DOI: 10.1080/22221751.2021.1873073 -
The Journal of Molecular Diagnostics :... Jun 2021
Topics: Biological Assay; COVID-19; Humans; Multiplex Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 33866002
DOI: 10.1016/j.jmoldx.2021.04.001 -
Biosensors Dec 2022Pathogenic bacteria are the leading causes of food-borne and water-borne infections, and one of the most serious public threats. Traditional bacterial detection... (Review)
Review
Pathogenic bacteria are the leading causes of food-borne and water-borne infections, and one of the most serious public threats. Traditional bacterial detection techniques, including plate culture, polymerase chain reaction, and enzyme-linked immunosorbent assay are time-consuming, while hindering precise therapy initiation. Thus, rapid detection of bacteria is of vital clinical importance in reducing the misuse of antibiotics. Among the most recently developed methods, the label-free optical approach is one of the most promising methods that is able to address this challenge due to its rapidity, simplicity, and relatively low-cost. This paper reviews optical methods such as surface-enhanced Raman scattering spectroscopy, surface plasmon resonance, and dark-field microscopic imaging techniques for the rapid detection of pathogenic bacteria in a label-free manner. The advantages and disadvantages of these label-free technologies for bacterial detection are summarized in order to promote their application for rapid bacterial detection in source-limited environments and for drug resistance assessments.
Topics: Bacteria; Surface Plasmon Resonance; Polymerase Chain Reaction; Enzyme-Linked Immunosorbent Assay; Spectrum Analysis, Raman
PubMed: 36551138
DOI: 10.3390/bios12121171 -
Molecules (Basel, Switzerland) Dec 2022Polymerase Chain Reaction (PCR) is one of the most common technologies used to produce millions of copies of targeted nucleic acid in vitro and has become an... (Review)
Review
Polymerase Chain Reaction (PCR) is one of the most common technologies used to produce millions of copies of targeted nucleic acid in vitro and has become an indispensable technique in molecular biology. However, it suffers from low efficiency and specificity problems, false positive results, and so on. Although many conditions can be optimized to increase PCR yield, such as the magnesium ion concentration, the DNA polymerases, the number of cycles, and so on, they are not all-purpose and the optimization can be case dependent. Nano-sized materials offer a possible solution to improve both the quality and productivity of PCR. In the last two decades, nanoparticles (NPs) have attracted significant attention and gradually penetrated the field of life sciences because of their unique chemical and physical properties, such as their large surface area and small size effect, which have greatly promoted developments in life science and technology. Additionally, PCR technology assisted by NPs (NanoPCR) such as gold NPs (Au NPs), quantum dots (QDs), and carbon nanotubes (CNTs), etc., have been developed to significantly improve the specificity, efficiency, and sensitivity of PCR and to accelerate the PCR reaction process. This review discusses the roles of different types of NPs used to enhance PCR and summarizes their possible mechanisms.
Topics: Metal Nanoparticles; Nanotubes, Carbon; Nanostructures; Polymerase Chain Reaction; Quantum Dots; Gold
PubMed: 36557991
DOI: 10.3390/molecules27248854 -
Biosensors Jan 2024Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease... (Review)
Review
Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.
Topics: Polymerase Chain Reaction; Microfluidics; DNA; RNA; Single-Cell Analysis
PubMed: 38391982
DOI: 10.3390/bios14020064