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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 -
Molecular Aspects of Medicine Jun 2024Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time... (Review)
Review
Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.
Topics: Nucleic Acid Denaturation; Humans; DNA; Polymerase Chain Reaction
PubMed: 38489863
DOI: 10.1016/j.mam.2024.101268 -
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 -
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 -
Methods in Molecular Biology (Clifton,... 2022DNA-encoded library (DEL) yields can be easily measured throughout the selection process using the quantitative polymerase chain reaction (qPCR) (Sannino A, Gabriele E,...
DNA-encoded library (DEL) yields can be easily measured throughout the selection process using the quantitative polymerase chain reaction (qPCR) (Sannino A, Gabriele E, Bigatti M, Mulatto S, Piazzi J, Scheuermann J, Neri D, Donckele EJ, Samain F, Chembiochem Eur J Chem Biol 20:955-962, 2019). Samples taken throughout the selection process are diluted prior to amplification and compared to standards of known DNA concentration. Here, I describe a general protocol using a double-stranded DNA binding dye for reaction monitoring. This allows the selection process to be assessed at each step prior to preparation for sequencing. The same method has additional applications in the practice of DEL technology.
Topics: DNA; Gene Library; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 36083552
DOI: 10.1007/978-1-0716-2545-3_17 -
Parasites & Vectors Nov 2022Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite... (Review)
Review
Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite of a female sand fly, Leishmania infections are increasing with human movement (due to international travel and war) as well as with shifts in vector habitat (due to climate change). Accurate diagnosis of the 20 or so species of Leishmania that infect humans can lead to the successful treatment of infections and, importantly, their prevention through modelling and intervention programs. A multitude of laboratory techniques for the detection of Leishmania have been developed over the past few decades, and although many have drawbacks, several of them show promise, particularly molecular methods like polymerase chain reaction. This review provides an overview of the methods available to diagnostic laboratories, from traditional techniques to the now-preferred molecular techniques, with an emphasis on polymerase chain reaction-based detection and typing methods.
Topics: Animals; Humans; Female; Leishmaniasis; Psychodidae; Leishmania; Polymerase Chain Reaction; Phlebotomus
PubMed: 36335408
DOI: 10.1186/s13071-022-05524-z -
Methods in Molecular Biology (Clifton,... 2023Polymerase chain reaction (PCR) has been a powerful molecular biology tool since the mid-1980s. Millions of copies of specific sequence regions of DNA can be generated...
Polymerase chain reaction (PCR) has been a powerful molecular biology tool since the mid-1980s. Millions of copies of specific sequence regions of DNA can be generated to allow the study of these regions. Fields that use this technology range from forensics to the experimental study of human biology. Standards for performing PCR and information tools to help design PCR protocols aid in successful implementation of PCR.
Topics: Humans; Taq Polymerase; Polymerase Chain Reaction; DNA
PubMed: 37041436
DOI: 10.1007/978-1-0716-2950-5_1 -
Malaria Journal Jul 2020Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease...
BACKGROUND
Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of P. knowlesi and P. vivax, and compare this method to nested PCR and qPCR.
METHODS
The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.
RESULTS
The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.
CONCLUSIONS
Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.
Topics: Diagnostic Tests, Routine; Humans; Malaysia; Plasmodium knowlesi; Plasmodium vivax; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 32650774
DOI: 10.1186/s12936-020-03314-5 -
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