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BioTechniques Oct 2020PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the... (Review)
Review
PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.
Topics: Animals; Betacoronavirus; COVID-19; COVID-19 Testing; Clinical Laboratory Techniques; Coronavirus Infections; Equipment Design; Humans; Microfluidic Analytical Techniques; Pandemics; Pneumonia, Viral; Point-of-Care Systems; Polymerase Chain Reaction; SARS-CoV-2
PubMed: 32815744
DOI: 10.2144/btn-2020-0057 -
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 -
Journal of Biomedical Science Jun 2023The global burden of invasive fungal infections (IFIs) has shown an upsurge in recent years due to the higher load of immunocompromised patients suffering from various... (Review)
Review
BACKGROUND
The global burden of invasive fungal infections (IFIs) has shown an upsurge in recent years due to the higher load of immunocompromised patients suffering from various diseases. The role of early and accurate diagnosis in the aggressive containment of the fungal infection at the initial stages becomes crucial thus, preventing the development of a life-threatening situation. With the changing demands of clinical mycology, the field of fungal diagnostics has evolved and come a long way from traditional methods of microscopy and culturing to more advanced non-culture-based tools. With the advent of more powerful approaches such as novel PCR assays, T2 Candida, microfluidic chip technology, next generation sequencing, new generation biosensors, nanotechnology-based tools, artificial intelligence-based models, the face of fungal diagnostics is constantly changing for the better. All these advances have been reviewed here giving the latest update to our readers in the most orderly flow.
MAIN TEXT
A detailed literature survey was conducted by the team followed by data collection, pertinent data extraction, in-depth analysis, and composing the various sub-sections and the final review. The review is unique in its kind as it discusses the advances in molecular methods; advances in serology-based methods; advances in biosensor technology; and advances in machine learning-based models, all under one roof. To the best of our knowledge, there has been no review covering all of these fields (especially biosensor technology and machine learning using artificial intelligence) with relevance to invasive fungal infections.
CONCLUSION
The review will undoubtedly assist in updating the scientific community's understanding of the most recent advancements that are on the horizon and that may be implemented as adjuncts to the traditional diagnostic algorithms.
Topics: Humans; Artificial Intelligence; Invasive Fungal Infections; Polymerase Chain Reaction
PubMed: 37337179
DOI: 10.1186/s12929-023-00926-2 -
BioTechniques Jul 2020Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids...
Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.
Topics: DNA; Emulsions; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Silicon Dioxide
PubMed: 32338528
DOI: 10.2144/btn-2019-0161 -
Iranian Biomedical Journal Nov 2020Human rotavirus (HRV) is the causative agent of severe gastroenteritis in children and responsible for two million hospitalizations and more than a half-million deaths...
BACKGROUND
Human rotavirus (HRV) is the causative agent of severe gastroenteritis in children and responsible for two million hospitalizations and more than a half-million deaths annually. Sequence characteristics of the gene segments encoding the VP7 and VP4 proteins are used for the genotype classification of rotavirus. A wide variety of molecular methods are available, mainly based on reverse transcription PCR for rapid, specific and sensitive genotyping of rotaviruses. This study describes an alternative real-time PCR assay for genotyping of rotavirus.
METHODS
The samples of stools studied in this research have been collected from patients referred to Children's Medical Centers, Tehran, Iran. Rotavirus detection and genotyping were performed using the RT-PCR and semi-nested RT-PCR, respectively. Samples were then genotyped with a new real-time PCR.
RESULTS
The real-time PCR was able to genotype all positive samples with a mean Ct of 28.2. Besides, a concordance rate of 100% was detected between real-time PCR and semi-nested RT-PCR.
CONCLUSION
In this study, the genotyping of rotavirus with real-time PCR showed that this method can provide several favorable features, including high sensitivity and specificity, and a wide dynamic range for rotavirus genotyping.
Topics: Child, Preschool; Genotyping Techniques; Humans; Limit of Detection; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Rotavirus; Rotavirus Infections
PubMed: 32660931
DOI: 10.29252/ibj.24.6.394 -
Cells Nov 2021Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring...
Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab')2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007-0.06%) when compared to the F(ab')2 antibody, CD19 protein and Protein L with 0.80% (range 0.47-1.58%), 0.65% (range 0.25-1.35%) and 0.73% (range 0.44-1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.
Topics: Antigens, CD19; Flow Cytometry; Humans; Immunotherapy, Adoptive; Indicators and Reagents; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 34831430
DOI: 10.3390/cells10113208 -
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