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BMC Research Notes May 2022The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The...
OBJECTIVE
The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA).
RESULTS
We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.
Topics: Adaptor Proteins, Signal Transducing; Cell-Free Nucleic Acids; Circulating Tumor DNA; DNA Methylation; Humans; Mesothelioma; Mesothelioma, Malignant; Polymerase Chain Reaction
PubMed: 35562749
DOI: 10.1186/s13104-022-06056-6 -
Research in Microbiology 2022Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a... (Review)
Review
Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a problematic issue. Fungal cultivation, the standard method that allows a proven diagnosis, has numerous disadvantages, as low sensitivity (only 50% of the patients present positive fungal cultures), and long growth time. These are factors that delay the patient's treatment and, consequently, lead to higher hospital costs. To improve the accuracy and quickness of fungal infections diagnosis, several new methodologies attempt to be implemented in clinical microbiology laboratories. Most of these innovative methods are independent of pathogen isolation, which means that the diagnosis goes from being considered proven to probable. In spite of the advantage of being culture-independent, the majority of the methods lack standardization. PCR-based methods are becoming more and more commonly used, which has earned them an important place in hospital laboratories. This can be perceived now, as PCR-based methodologies have proved to be an essential tool fighting against the COVID-19 pandemic. This review aims to go through the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as "gold standard", to the molecular methods currently used, and finally mentioning some of the more futuristic approaches.
Topics: COVID-19; Humans; Mycoses; Pandemics; Polymerase Chain Reaction
PubMed: 34863883
DOI: 10.1016/j.resmic.2021.103915 -
Clinical Infectious Diseases : An... Dec 2021
Topics: COVID-19; Communicable Diseases; Diagnostic Tests, Routine; Humans; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 33277652
DOI: 10.1093/cid/ciaa1819 -
Surgical Infections Oct 2020Currently, one of the most pressing problems in the field of orthopedic surgery is peri-prosthetic joint infection [PJI]. While there are numerous ways to detect PJI,... (Review)
Review
Currently, one of the most pressing problems in the field of orthopedic surgery is peri-prosthetic joint infection [PJI]. While there are numerous ways to detect PJI, current clinical detection methods differ across institutions and have varying criteria and protocols. Some of these methods include the Modified Musculoskeletal Infection Society system, culturing, polymerase chain reaction, the determination of the presence of certain biomarkers, testing for the presence of alpha defensin peptides, and leukocyte level testing. This review summarizes the most recent publications in the field of PJI detection to highlight current strengths as well as provide future directions to find the system for the quickest, cost-effective, and most accurate way to diagnose these types of infections. The results of this literature review suggest that, while each method of diagnosis has its advantages, each has various drawbacks as well. Current methods can be expensive, take days to weeks to complete, be prone to contamination, and can produce ambiguous results. The findings in this review emphasize the need for a more comprehensive and accurate system for diagnosing PJI. In addition, the specific comparison of advantages and drawbacks can be useful for researchers and clinicians with goals of creating new diagnostic tests for PJIs, as well as in clinical scenarios to determine the correct treatment for patients.
Topics: Biomarkers; Blood Culture; Humans; Leukocyte Count; Polymerase Chain Reaction; Prosthesis-Related Infections; alpha-Defensins
PubMed: 32043924
DOI: 10.1089/sur.2019.314 -
PLoS Neglected Tropical Diseases Jun 2023With the development of domestic animal husbandry, the spread of brucellosis has accelerated, and the scope of the epidemic has expanded. The timely and accurate...
BACKGROUND
With the development of domestic animal husbandry, the spread of brucellosis has accelerated, and the scope of the epidemic has expanded. The timely and accurate diagnosis of human brucellosis continues to challenge clinicians in endemic areas. Droplet digital PCR (ddPCR) technology can quickly and accurately determine DNA load in samples, providing laboratory evidence for diagnosis, prognosis and management of brucellosis patients. In this study, a ddPCR method was established to accurately quantify Brucella DNA load in whole blood samples, and its diagnostic, prognostic, and therapeutic value for human brucellosis was evaluated.
METHODS
Annealing temperature, primers, and probe targeting the Brucella bcsp31 gene were optimised, and the sensitivity, specificity and repeatability of the ddPCR assay were assessed using 94 whole blood samples from 61 confirmed and 33 suspected cases. Results were compared with those of quantitative PCR (qPCR). Nine follow-up brucellosis patients were also analysed by the two methods after 2 and 6 months of treatment.
RESULTS
Optimal primer and probe concentrations were 800 nmol/L and 400 nmol/L, respectively, and the optimal annealing temperature was 55.3 °C. The ddPCR results showed that the limit of detection was 1.87 copies per reaction, with high repeatability. The positive rates for ddPCR and qPCR were 88.5% and 75.4% among 61 serum agglutination test (SAT) positive patients. In addition, 57.6% (19/33) of suspected sero-negative samples were positive by ddPCR, but only 36.3% (12/33) were positive by qPCR. Analysis of nine post-therapy follow-up brucellosis patients revealed that the Brucella DNA load in the whole blood samples decreased after 2 and 6 months of treatment, and was slightly increased following relapse and continuous exposure.
CONCLUSION
The ddPCR assay showed good accuracy for whole blood samples, and could be a potential diagnostic and prognostic tool for detecting Brucella.
Topics: Animals; Humans; Brucella; Sensitivity and Specificity; Polymerase Chain Reaction; Brucellosis; Serum; Real-Time Polymerase Chain Reaction
PubMed: 37267228
DOI: 10.1371/journal.pntd.0011367 -
Scientific Reports Nov 2023Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3...
Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2-3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50-60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.
Topics: Animals; Humans; RNA; Polymerase Chain Reaction; RNA, Messenger; Cell Line; Biological Assay; Real-Time Polymerase Chain Reaction; Mammals
PubMed: 37923834
DOI: 10.1038/s41598-023-46314-6 -
International Journal of Molecular... Jan 2023Low-level tumor somatic DNA mutations in tissue and liquid biopsies obtained from cancer patients can have profound implications for development of metastasis,... (Review)
Review
Low-level tumor somatic DNA mutations in tissue and liquid biopsies obtained from cancer patients can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up, or early cancer detection. Unless detected, such low-frequency DNA alterations can misinform patient management decisions or become missed opportunities for personalized medicine. Next-generation sequencing technologies and digital-PCR can resolve low-level mutations but require access to specialized instrumentation, time, and resources. Enzymatic-based approaches to detection of low-level mutations provide a simple, straightforward, and affordable alternative to enrich and detect such alterations and is broadly available to low-resource laboratory settings. This review summarizes the traditional uses of enzymatic mutation detection and describes the latest exciting developments, potential, and applications with specific reference to the field of liquid biopsy in cancer.
Topics: Humans; Neoplasms; Liquid Biopsy; DNA, Neoplasm; Mutation; Polymerase Chain Reaction; High-Throughput Nucleotide Sequencing; Biomarkers, Tumor
PubMed: 36674433
DOI: 10.3390/ijms24020923 -
Molecular Aspects of Medicine Apr 2024The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and... (Review)
Review
The quantitative polymerase chain reaction (qPCR) is fundamental to molecular biology. It is not just a laboratory technique, qPCR is a bridge between research and clinical practice. Its theoretical foundations guide the design of experiments, while its practical implications extend to diagnostics, treatment, and research advancements in the life sciences, human and veterinary medicine, agriculture, and forensics. However, the accuracy, reliability and reproducibility of qPCR data face challenges arising from various factors associated with experimental design, execution, data analysis and inadequate reporting details. Addressing these concerns, the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have emerged as a cohesive framework offering a standardised set of recommendations that describe the essential information required for assessing qPCR experiments. By emphasising the importance of methodological rigour, the MIQE guidelines have made a major contribution to improving the trustworthiness, consistency, and transparency of many published qPCR results. However, major challenges related to awareness, resources, and publication pressures continue to affect their consistent application.
Topics: Humans; Reproducibility of Results; Real-Time Polymerase Chain Reaction
PubMed: 38290180
DOI: 10.1016/j.mam.2024.101249 -
Talanta Mar 2021In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important...
In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important technologies for RNA detection and disease diagnosis. To achieve high quality assurance, appropriate positive and negative controls are critical for disease detection using RT-PCR kits. In this study, we have found that commercial kits often adopt DNAs instead of RNAs as the positive controls, which can't report the kit problems in reverse transcription, thereby increasing risk of the false negative results when testing patient samples. To face the challenge, we have proposed and developed the chemically modified RNAs, such as phosphoroselenaote and phosphorothioate RNAs (Se-RNA and S-RNA), as the controls. We have found that while demonstrating the high thermostability, biostability, chemostability and exclusivity (or specificity), both Se-RNA and S-RNA can be fine templates for reverse transcription, indicating their potentials as both positive and negative controls for RT-PCR kits.
Topics: COVID-19; COVID-19 Nucleic Acid Testing; DNA, Viral; False Negative Reactions; Humans; RNA Stability; RNA, Viral; Reagent Kits, Diagnostic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 33379066
DOI: 10.1016/j.talanta.2020.121850 -
International Journal of Molecular... Jan 2022Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell...
Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.
Topics: B-Cell Maturation Antigen; Biomarkers; Flow Cytometry; Humans; Immunophenotyping; Immunotherapy, Adoptive; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Receptors, Chimeric Antigen; Reproducibility of Results; Sensitivity and Specificity; T-Lymphocytes
PubMed: 35055086
DOI: 10.3390/ijms23020903