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Mikrochimica Acta Jun 2023Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however,...
Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources.
Topics: Hydrogels; Capsules; Polymerase Chain Reaction; Nucleic Acids
PubMed: 37261544
DOI: 10.1007/s00604-023-05827-7 -
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 -
Journal of Veterinary Internal Medicine May 2018Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive... (Review)
Review
Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture-based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.
Topics: Animals; Cattle; Cattle Diseases; Milk; Mycoplasma; Mycoplasma Infections; Mycoplasma bovis; Polymerase Chain Reaction; Serologic Tests
PubMed: 29671903
DOI: 10.1111/jvim.15135 -
Lakartidningen Mar 2018
Topics: Diagnostic Test Approval; Humans; Molecular Diagnostic Techniques; Polymerase Chain Reaction; Spectrometry, Mass, Electrospray Ionization
PubMed: 29533414
DOI: No ID Found -
International Journal of Environmental... Apr 2022Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health... (Review)
Review
Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health problems, such as waterborne outbreaks, which have caused huge economic and social losses. In this context, the prompt detection of microbial contamination becomes essential to enable early warning and timely reaction with proper interventions. Recently, molecular diagnostics have been increasingly employed for the rapid and robust assessment of microbial water quality implicated by various microbial pollutants, e.g., waterborne pathogens and antibiotic-resistance genes (ARGs), imposing the most critical health threats to humans and the environment. Continuous technological advances have led to constant improvements and expansions of molecular methods, such as conventional end-point PCR, DNA microarray, real-time quantitative PCR (qPCR), multiplex qPCR (mqPCR), loop-mediated isothermal amplification (LAMP), digital droplet PCR (ddPCR), and high-throughput next-generation DNA sequencing (HT-NGS). These state-of-the-art molecular approaches largely facilitate the surveillance of microbial water quality in diverse aquatic systems and wastewater. This review provides an up-to-date overview of the advancement of the key molecular tools frequently employed for microbial water quality assessment, with future perspectives on their applications.
Topics: Drug Resistance, Microbial; Multiplex Polymerase Chain Reaction; Pathology, Molecular; Real-Time Polymerase Chain Reaction; Water Quality
PubMed: 35564522
DOI: 10.3390/ijerph19095128 -
Advanced Healthcare Materials Jan 2016Recent outbreaks of deadly infectious diseases, such as Ebola and Middle East respiratory syndrome coronavirus, have motivated the research for accurate, rapid...
Recent outbreaks of deadly infectious diseases, such as Ebola and Middle East respiratory syndrome coronavirus, have motivated the research for accurate, rapid diagnostics that can be administered at the point of care. Nucleic acid biomarkers for these diseases can be amplified and quantified via polymerase chain reaction (PCR). In order to solve the problems of conventional PCR--speed, uniform heating and cooling, and massive metal heating blocks--an innovative optofluidic cavity PCR method using light-emitting diodes (LEDs) is accomplished. Using this device, 30 thermal cycles between 94 °C and 68 °C can be accomplished in 4 min for 1.3 μL (10 min for 10 μL). Simulation results show that temperature differences across the 750 μm thick cavity are less than 2 °C and 0.2 °C, respectively, at 94 °C and 68 °C. Nucleic acid concentrations as low as 10(-8) ng μL(-1) (2 DNA copies per μL) can be amplified with 40 PCR thermal cycles. This simple, ultrafast, precise, robust, and low-cost optofluidic cavity PCR is favorable for advanced molecular diagnostics and precision medicine. It is especially important for the development of lightweight, point-of-care devices for use in both developing and developed countries.
Topics: Computer Simulation; Electronics; Gold; Humans; Optical Phenomena; Polymerase Chain Reaction; Temperature
PubMed: 26592501
DOI: 10.1002/adhm.201500708 -
Molecular and Cellular Biochemistry Jul 2023Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore... (Review)
Review
Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.
Topics: Ligases; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Mutation; Technology; Nucleic Acids
PubMed: 36441353
DOI: 10.1007/s11010-022-04615-w -
Cytotherapy Jan 2023Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be...
BACKGROUND AIMS
Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct.
METHODS
The authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer-probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct.
RESULTS
ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors' assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities.
CONCLUSIONS
The authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test-retest reliability, and that specific assays can be developed for detection of unique constructs.
Topics: Humans; Reproducibility of Results; Receptors, Chimeric Antigen; DNA Copy Number Variations; T-Lymphocytes; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 36253252
DOI: 10.1016/j.jcyt.2022.09.004 -
BioTechniques Jul 2019
Topics: Animals; Equipment Design; Humans; Miniaturization; Polymerase Chain Reaction; Time Factors
PubMed: 31238703
DOI: 10.2144/btn-2019-0076 -
Molecular Biotechnology Apr 2022The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent... (Review)
Review
The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.
Topics: Biosensing Techniques; COVID-19 Testing; CRISPR-Cas Systems; Electrochemical Techniques; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Molecular Diagnostic Techniques; Neutralization Tests; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; SARS-CoV-2; Surface Plasmon Resonance
PubMed: 34655396
DOI: 10.1007/s12033-021-00417-5