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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 -
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 -
Molecular Biology Reports Nov 2023Plant pathogens cause severe losses to agricultural yield worldwide. Tracking plant health and early disease detection is important to reduce the disease spread and thus... (Review)
Review
Plant pathogens cause severe losses to agricultural yield worldwide. Tracking plant health and early disease detection is important to reduce the disease spread and thus economic loss. Though visual scouting has been practiced from former times, detection of asymptomatic disease conditions is difficult. So, DNA-based and serological methods gained importance in plant disease detection. The progress in advanced technologies challenges the development of rapid, non-invasive, and on-field detection techniques such as spectroscopy. This review highlights various direct and indirect ways of detecting plant diseases like Enzyme-linked immunosorbent assay, Lateral flow assays, Polymerase chain reaction, spectroscopic techniques and biosensors. Although these techniques are sensitive and pathogen-specific, they are more laborious and time-intensive. As a consequence, a lot of interest is gained in in-field adaptable point-of-care devices with artificial intelligence-assisted pathogen detection at an early stage. More recently computer-aided techniques like neural networks are gaining significance in plant disease detection by image processing. In addition, a concise report on the latest progress achieved in plant disease detection techniques is provided.
Topics: Artificial Intelligence; Plants; Polymerase Chain Reaction; Plant Diseases
PubMed: 37823933
DOI: 10.1007/s11033-023-08838-y -
International Journal of Biological... Oct 2018Polymerase chain reaction (PCR) has become a popular diagnosis and research technique in dentistry. Several studies show that high sensitivity and specificity of PCR... (Review)
Review
Polymerase chain reaction (PCR) has become a popular diagnosis and research technique in dentistry. Several studies show that high sensitivity and specificity of PCR method allows it to be as a precise, efficient, and rapid method for detection, identification, and quantification of microorganisms. Several genetic polymorphisms along with immune and inflammatory markers can be determined; providing the most accurate perception into the mechanisms underlying the dental and periodontal disease. This review paper discusses the application of PCR as a diagnostic molecular technique in periodontology, endodontic infections, implant-related and peri-implantitis infection, identification of immune and inflammatory markers, and genetic polymorphisms. Moreover, it reviews the application of next generation sequencing and microarray technologies in dentistry. The manuscript was prepared mainly using PubMed and MEDLINE.
Topics: Dentistry; High-Throughput Nucleotide Sequencing; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction
PubMed: 29778881
DOI: 10.1016/j.ijbiomac.2018.05.085 -
Molecular Diagnosis & Therapy Oct 2016Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has... (Review)
Review
Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.
Topics: Humans; Polymerase Chain Reaction; Reproducibility of Results; Research; Sensitivity and Specificity; Virology; Virus Diseases; Viruses
PubMed: 27351921
DOI: 10.1007/s40291-016-0224-1 -
Cold Spring Harbor Protocols May 2018The purpose of hot start polymerase chain reaction (PCR) is to optimize the yield of the desired amplified product in PCRs and, simultaneously, to suppress nonspecific...
The purpose of hot start polymerase chain reaction (PCR) is to optimize the yield of the desired amplified product in PCRs and, simultaneously, to suppress nonspecific amplification and formation of primer dimers. This is achieved by withholding an essential component of the PCR-the DNA polymerase, or the primers, for example-until the reaction mixture has been heated to a temperature that inhibits hybridization of primers to one another or to nonspecific regions of the template.
Topics: DNA; Hot Temperature; Indicators and Reagents; Polymerase Chain Reaction
PubMed: 29717052
DOI: 10.1101/pdb.prot095125 -
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 -
Methods in Molecular Biology (Clifton,... 2023Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and...
Droplet digital polymerase chain reaction (ddPCR) is a new quantitative PCR method based on water-oil emulsion droplet technology. ddPCR enables highly sensitive and accurate quantification of nucleic acid molecules, especially when their copy numbers are low. In ddPCR, a sample is fractionated into ~20,000 droplets, and every nanoliter-sized droplet undergoes PCR amplification of the target molecule. The fluorescence signals of droplets are then recorded by an automated droplet reader. Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that are ubiquitously expressed in animals and plants. CircRNAs are promising as biomarkers for cancer diagnosis and prognosis and as therapeutic targets or agents to inhibit oncogenic microRNAs or proteins (Kristensen LS, Jakobsen T, Hager H, Kjems J, Nat Rev Clin Oncol 19:188-206, 2022). In this chapter, the procedures for the quantitation of a circRNA in single pancreatic cancer cells using ddPCR are described.
Topics: Single-Cell Analysis; RNA, Circular; Polymerase Chain Reaction; Pancreatic Neoplasms; Cell Line, Tumor; Biomarkers, Tumor; Humans
PubMed: 37430054
DOI: 10.1007/978-1-0716-3323-6_13 -
Animal Science Journal = Nihon Chikusan... 2021Halal products are growing in consumer markets worldwide, and pork meat is classified as non-halal. Manufacturers of processed foods and products must ensure that their...
Halal products are growing in consumer markets worldwide, and pork meat is classified as non-halal. Manufacturers of processed foods and products must ensure that their products follow Islamic dietary law because pork is prohibited for Muslims. Droplet digital polymerase chain reaction (PCR) (ddPCR) is a novel method for identifying pig species and quantifying pork products. This experiment aimed to investigate pork species and establish the proportion of pork in meat products using the mitochondrial cytochrome b gene (CYTB). The study found that the correlation coefficient between the meat weight and DNA concentration of pork was 0.997, and the correlation coefficient between the DNA concentration and the target DNA copy number of pork was 0.998. The accuracy of the ddPCR assay was verified using a sample of a known proportion of pork, and it was revealed that this method is highly precise in quantifying pork products. Nine products contained an undeclared meat proportion (90%). The limit of detection for pork was 0.0001 ng. The analysis indicated that the ddPCR assay has high accuracy and sensitivity for quantifying pork products. Therefore, the predictive model can be used in routine laboratories for quality assurance of halal food products.
Topics: Animals; DNA; Meat Products; Polymerase Chain Reaction; Pork Meat; Real-Time Polymerase Chain Reaction; Red Meat; Swine
PubMed: 34318546
DOI: 10.1111/asj.13595