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Methods in Molecular Biology (Clifton,... 2021Semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) is a simple and specific method for quantitative RNA in recent years. The relative...
Semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) is a simple and specific method for quantitative RNA in recent years. The relative quantity of a specific mRNA in the samples can be inferred by reverse transcription of mRNA into cDNA, and PCR amplification and determination of the quantity of PCR products. The semi-quantitative analysis is carried out under a fixed number of PCR cycles, and the total RNA concentration is kept in the exponential phase of the PCR. The method is to use a housekeeping gene (usually actin, GAPDH, and EF1α) as a reference standard in treated and control organisms to observe the expression of the interested genes (upregulated or downregulated) in toxicology. In this chapter, we describe a step-by-step method for determining the differential regulation of target genes in organisms exposed to environmental pollutants.
Topics: Animals; Environmental Pollutants; Humans; Polymerase Chain Reaction; RNA; Toxicity Tests; Transcription, Genetic
PubMed: 34097263
DOI: 10.1007/978-1-0716-1514-0_7 -
Methods in Molecular Biology (Clifton,... 2023Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for...
Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for speed; sensitivity, specificity, and yield are equivalent or better than conventional PCR. What is required (and not widely available) is rapid, accurate control of reaction temperature during cycling. Specificity improves with cycling speed, and efficiency can be maintained by increasing polymerase and primer concentrations. Speed is aided by simplicity, dyes that stain double-stranded DNA are less expensive than probes, and one of the simplest polymerases, the deletion mutant KlenTaq, is used throughout. Rapid amplification can be coupled with endpoint melting analysis to verify product identity. Instead of commercial master mixes, detailed formulations for reagents and master mixes compatible with rapid cycle and extreme PCR are described.
Topics: Polymerase Chain Reaction; DNA; Temperature
PubMed: 37041449
DOI: 10.1007/978-1-0716-2950-5_14 -
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 -
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 -
Methods in Molecular Biology (Clifton,... 2023Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction...
Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction (PCR) with two competing allele-specific primers and a SNP specific common reverse primer. Sequence information flanking SNP of interest and fluorescent plate reader for end-point measurement or qPCR machine for real time measurement are required for the execution of the Amplifluor assay. In this chapter, the principle and working protocol of the Amplifluor assay based on end-point fluorescence detection of SNP allele is presented with an example.
Topics: Genotype; Polymorphism, Single Nucleotide; Polymerase Chain Reaction; DNA Primers; Alleles
PubMed: 36781643
DOI: 10.1007/978-1-0716-3024-2_13 -
Analytical Biochemistry Mar 2022Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed....
Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 μl reaction volume, 2) the supply of external temperature 145°С for the denaturation zone and 0°С for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.
Topics: COVID-19; COVID-19 Nucleic Acid Testing; Convection; Humans; Polymerase Chain Reaction; RNA, Viral; SARS-CoV-2; Temperature; Time Factors
PubMed: 35074320
DOI: 10.1016/j.ab.2022.114565 -
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 -
ACS Applied Materials & Interfaces Jul 2022A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic...
A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic testing involving small amounts of samples. The use of a free enzyme in conventional DNA amplification techniques, such as the polymerase chain reaction (PCR), frequently causes side reactions (i.e., nonspecific DNA amplification) when ≤10 substrate DNA molecules are present, thereby preventing selective amplification of the target DNA. To address this issue, we have developed a novel DNA amplification system, mesoporous silica-enhanced PCR (MSE-PCR), which involves the immobilization of a thermostable DNA polymerase from (KOD DNA polymerase) into highly ordered nanopores of the mesoporous silica to control the reaction environment around the enzyme. In the MSE-PCR system using immobilized KOD DNA polymerase, such nonspecific DNA amplification was remarkably inhibited under the same conditions. Furthermore, the optimization of mesoporous silica pore sizes enabled selective and efficient DNA amplification from DNA substrates at the single-molecule level, i.e., one ten-thousandth of the amount of substrate DNA required for a DNA amplification reaction using a free enzyme. The results obtained in this study have shown that the nanopores of mesoporous silica can inhibit nonspecific reactions in DNA amplification, thereby considerably improving the specificity and sensitivity of the DNA polymerase reaction.
Topics: DNA; DNA-Directed DNA Polymerase; Enzymes, Immobilized; Immobilized Nucleic Acids; Polymerase Chain Reaction; Silicon Dioxide
PubMed: 35700238
DOI: 10.1021/acsami.2c01992 -
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