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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 -
BMC Ophthalmology Oct 2016To study the value and safety of aqueous humor polymerase chain reaction (PCR) analysis for Herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr virus and...
BACKGROUND
To study the value and safety of aqueous humor polymerase chain reaction (PCR) analysis for Herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr virus and Toxoplasma gondii in patients with uveitis.
METHODS
Records of 45 consecutive patients with anterior and posterior uveitis who underwent AC paracentesis with PCR were reviewed. The main outcome measure was frequency of PCR positivity. Secondary outcomes were alteration of treatment, safety of paracentesis, and correlation of keratitic precipitates with PCR positivity, RESULTS: The overall PCR positivity was 48.9 % (22/45). Therapy was changed because of the PCR results in 14/45 patients (37.7 %). One patient experienced a paracentesis related complication (1/45, 2.2 %) without long-term sequelae.
CONCLUSION
Aqueous PCR altered the diagnosis and treatment in over a third of our patients and was relatively safe. Aqueous PCR should be considered for uveitis of atypical clinical appearance, recurrent severe uveitis of uncertain etiology, and therapy refractory cases.
Topics: Adult; Aged; Aqueous Humor; Diagnostic Techniques, Ophthalmological; Eye Infections, Viral; Female; Humans; Male; Middle Aged; Polymerase Chain Reaction; Toxoplasmosis; Uveitis; Young Adult
PubMed: 27793120
DOI: 10.1186/s12886-016-0369-z -
Sheng Wu Gong Cheng Xue Bao = Chinese... Feb 2017Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated... (Review)
Review
Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.
Topics: DNA; DNA Copy Number Variations; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction
PubMed: 28956373
DOI: 10.13345/j.cjb.160269 -
Veterinary Pathology Jul 2017Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be...
Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.
Topics: Animals; Cat Diseases; Cats; Cattle; Cattle Diseases; DNA, Fungal; Dog Diseases; Dogs; Horse Diseases; Horses; Mycoses; Paraffin Embedding; Polymerase Chain Reaction; Sequence Alignment
PubMed: 28346123
DOI: 10.1177/0300985817698207 -
Biotechnology and Bioengineering Sep 2018We demonstrate the integration of DNA amplification and detection functionalities developed on a lab-on-a-chip microdevice utilizing solid-phase polymerase chain...
We demonstrate the integration of DNA amplification and detection functionalities developed on a lab-on-a-chip microdevice utilizing solid-phase polymerase chain reaction (SP-PCR) for point-of-need (PON) DNA analyses. First, the polycarbonate microdevice was fabricated by thermal bonding to contain microchambers as reservoirs for performing SP-PCR. Next, the microchambers were subsequently modified with polyethyleneimine and glutaraldehyde for immobilizing amine-modified forward primers. During SP-PCR, the immobilized forward primers and freely diffusing fluorescence-labeled reverse primers cooperated to generate target amplicons, which remained covalently attached to the microchambers for the fluorescence detection. The SP-PCR microdevice was used for the direct identifications of two widely detected foodborne pathogens, namely Salmonella spp. and Staphylococcus aureus, and an alga causing harmful algal blooms annually in South Korea, Cochlodinium polykrikoides. The SP-PCR microdevice would be versatilely applied in PON testing as a universal platform for the fast identification of foodborne pathogens and environmentally threatening biogenic targets.
Topics: Dinoflagellida; Lab-On-A-Chip Devices; Microbiological Techniques; Molecular Diagnostic Techniques; Point-of-Care Systems; Polymerase Chain Reaction; Salmonella; Staphylococcus aureus; Time Factors
PubMed: 29777597
DOI: 10.1002/bit.26734 -
Fa Yi Xue Za Zhi Oct 2017Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a convenient and highly efficient method for the detection of mRNA in tissues or... (Review)
Review
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a convenient and highly efficient method for the detection of mRNA in tissues or body fluid samples. It has the characteristics of easy operation, high sensitivity and specificity, etc. With a wide application in medicine, biology and other fields, RT-qPCR technique has made some progresses in the research field of forensic pathology. This paper reviews the application value of RT-qPCR in the study of forensic pathology and current situation, as well as the research progress at home and abroad reviews. It also summarizes the notes of samples extraction, RT-qPCR experiments and data processing, which aims to provide reference for the forensic research and its application.
Topics: Forensic Genetics; Forensic Pathology; Humans; RNA, Messenger; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 29275561
DOI: 10.3969/j.issn.1004-5619.2017.05.017 -
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 -
ACS Synthetic Biology Dec 2015The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years, much work has been carried out using a...
The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years, much work has been carried out using a range of biomolecular chassis including α-helical coiled coils, β-sheet amyloids and even viral particles. In this work, we show how hybrid bionanoparticles can be produced from a viral M13 bacteriophage scaffold through conjugation with DNA primers that can template a polymerase chain reaction (PCR). This unprecedented example of a PCR on a virus particle has been studied by flow aligned linear dichroism spectroscopy, which gives information on the structure of the product as well as a new protototype methodology for DNA detection. We propose that this demonstration of PCR on the surface of a bionanoparticle is a useful addition to ways in which hybrid assemblies may be constructed using synthetic biology.
Topics: Bacteriophage M13; DNA Primers; Nanoparticles; Polymerase Chain Reaction
PubMed: 26046486
DOI: 10.1021/acssynbio.5b00034 -
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