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Archives of Virology Jan 2019The red-crowned crane is one of the rarest crane species, and its population is decreasing due to loss of habitat, poisoning, and infections. Using a viral metagenomics...
The red-crowned crane is one of the rarest crane species, and its population is decreasing due to loss of habitat, poisoning, and infections. Using a viral metagenomics approach, we analyzed the virome of feces from wild and captive red-crowned cranes, which were pooled separately. Vertebrate viruses belonging to the families Picornaviridae, Parvoviridae, Circoviridae, and Caliciviridae were detected. Among the members of the family Picornaviridae, we found three that appear to represent new genera. Six nearly complete genomes from members of the family Parvoviridae were also obtained, including four new members of the proposed genus "Chapparvovirus", and two members of the genus Aveparvovirus. Six small circular DNA genomes were also characterized. One nearly complete genome showing a low level of sequence identity to caliciviruses was also characterized. Numerous viruses believed to infect insects, plants, and crustaceans were also identified, which were probably derived from the diet of red-crowned cranes. This study increases our understanding of the enteric virome of red-crowned cranes and provides a baseline for comparison to those of other birds or following disease outbreaks.
Topics: Animals; Birds; Feces; Genome, Viral; Metagenome; Phylogeny; Polymerase Chain Reaction; Viruses
PubMed: 30225519
DOI: 10.1007/s00705-018-4037-x -
Virologica Sinica Jun 2018The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological...
Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus.
The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold (Ct) value was linear from 10 to 10 copy/μL, with a standard curve R of 0.999 and amplification efficiency of 92.203%; however, a concentration as low as 1 copy/μL could not be detected. In comparison with RT-qPCR, the ddPCR method resulted in a linear range of 10-10 copy/μL and was able to detect concentrations as low as 1 copy/μL. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples (above 10 copy/μL), while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.
Topics: Humans; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Zika Virus
PubMed: 29931514
DOI: 10.1007/s12250-018-0037-y -
Scientific Reports Mar 2017Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to 'stitch' individual pieces of DNA...
Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to 'stitch' individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user's input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html.
Topics: DNA Primers; Polymerase Chain Reaction; Software; Web Browser
PubMed: 28358011
DOI: 10.1038/srep45349 -
Scientific Reports Mar 2017Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard...
Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.
Topics: Calibration; Humans; Monte Carlo Method; Real-Time Polymerase Chain Reaction; Reproducibility of Results
PubMed: 28327545
DOI: 10.1038/srep44854 -
The Journal of Heart and Lung... Mar 2023Shortage of organ donors is an ongoing limiting factor in lung transplantation (LT). Despite increasing prevalence of asymptomatic COVID-19 infection, positive COVID-19...
Shortage of organ donors is an ongoing limiting factor in lung transplantation (LT). Despite increasing prevalence of asymptomatic COVID-19 infection, positive COVID-19 testing from a potential donor remains a contraindication at many LT centers. In this report, we present the outcomes of LT utilizing an algorithm based on donor clinical presentation, and COVID-19 real-time reverse transcription polymerase chain reaction (RT-PCR) with cycle threshold (Ct) values evaluation. The Ct value threshold for organ acceptance was >35. A total of 8 COVID-positive donors were included. No donor-to-recipient transmissions of COVID-19 were observed. Short-term outcomes were comparable to those reported in pre-COVID literature. Survival-to-date is 100% with median POD of 161 days. Our findings support the safety and efficacy of utilizing our algorithm including Ct value threshold for selection of donors with incidental COVID-19 positive testing.
Topics: Humans; COVID-19; COVID-19 Testing; Tissue Donors; Lung; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 36624019
DOI: 10.1016/j.healun.2022.12.016 -
Acta Veterinaria Hungarica Mar 2018Heteroduplex polymerase chain reaction for antigen receptor rearrangements (hPARR) was developed to monitor minimal residual disease (MRD) in canine B- and T-cell...
Heteroduplex polymerase chain reaction for antigen receptor rearrangements (hPARR) was developed to monitor minimal residual disease (MRD) in canine B- and T-cell lymphomas treated with the modified L-COP or L-CHOP protocol. Thirty-five dogs were recruited in this study and their neoplastic lineages were determined by immunophenotyping with Pax5 and CD3. Peripheral blood leukocytes were collected prior to and during chemotherapy in weeks 4, 9 and 13 to detect MRD by hPARR. Twenty-eight dogs (80%) had B-cell lymphoma while seven dogs (20%) had T-cell lymphoma. A monoclonal band was detected in 11 cases that showed complete or partial remission before tumour relapse and no response to the current treatment without statistical difference in clinical outcomes; however, the treatment response had an association with the MRD result (P < 0.05). Modified L-CHOP prolonged median progression-free survival as compared to modified L-COP (215 days vs. 93 days; P < 0.05). Substage b had shorter progression-free survival than substage a (90 days vs. 215 days; P < 0.05). Clinical stage III affected median overall survival time when compared to clinical stages IV and V (432, 173 and 118 days, respectively; P < 0.05). hPARR could be used for screening refractory lymphoma together with lymph node measurement in routine clinical cases.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Dog Diseases; Dogs; Lymphoma; Polymerase Chain Reaction
PubMed: 29580085
DOI: 10.1556/004.2018.007 -
Poultry Science Feb 2023Goose astrovirus (GoAstV), goose parvovirus (GPV), and goose circovirus (GoCV) infections have similar symptoms, such as severe diarrhea, and cause serious economic...
Goose astrovirus (GoAstV), goose parvovirus (GPV), and goose circovirus (GoCV) infections have similar symptoms, such as severe diarrhea, and cause serious economic losses to the goose industry globally. Therefore, it is necessary to develop a rapid and accurate method for the differential diagnosis of the 3 viruses. In this study, a TaqMan probe-based multiplex reverse transcription-qualitative polymerase chain reaction (RT-qPCR) method was established and optimized for simultaneous detection of the three viruses. Three pairs of specific primers and probes were designed considering the conserved sequences of ORF2, VP3, and Rep of GoAstV, GPV, and GoCV, respectively. Singleplex real-time RT-qPCR detected a minimum of 10 copies of these genes, while multiplex real-time RT-qPCR detected a minimum of 100 copies. The correlation coefficients exceeded 0.99, and the amplification efficiency was 80 to 100%. The assay had high sensitivity, specificity, and repeatability. In 85 tissue samples, GoAstV and GPV were the main pathogens and demonstrated co-infection. This assay provides a rapid, efficient, specific, and sensitive tool for the detection of GoAstV, GPV, and GoCV. This can facilitate disease management and epidemiological surveillance.
Topics: Animals; Chickens; Geese; Parvovirinae; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Taq Polymerase
PubMed: 36565640
DOI: 10.1016/j.psj.2022.102396 -
Journal of Clinical Laboratory Analysis Jan 2018Methylenetetrahydrofolate reductase (MTHFR; NM_005957.4) is the key enzyme for folate metabolism which plays in DNA biosynthesis and the epigenetic process of DNA...
A duplex polymerase chain reaction-restriction fragment length polymorphism for rapid screening of methylenetetrahydrofolate reductase gene variants: Genotyping in acute leukemia.
BACKGROUND
Methylenetetrahydrofolate reductase (MTHFR; NM_005957.4) is the key enzyme for folate metabolism which plays in DNA biosynthesis and the epigenetic process of DNA methylation. MTHFR gene polymorphisms, the c. 677C>T and c. 1298A>C have been implicated as risk factors for several types of cancers as the acute leukemia.
AIM
We have optimized a duplex polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) for the simultaneous detection of both variants in acute leukemia patients, from Tunisia.
METHODS
Genomic DNA was extracted from EDTA-anticoagulant blood samples from a total of 50 patients suffering from acute leukemia (AL). After DNA extraction, the polymerase chain reaction using specific primers, designed using Primer 3 Software. Restriction Fragment Length Polymorphism (RFLP) was performed in two separate tubes followed by agarose gel electrophoresis.
CONCLUSION
This new method has proved to be a rapid, simple, and reliable method that should facilitate high throughput genotyping of MTHFR polymorphisms in acute leukemia.
Topics: Genotyping Techniques; Humans; Leukemia; Methylenetetrahydrofolate Reductase (NADPH2); Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length
PubMed: 28374953
DOI: 10.1002/jcla.22198 -
Scientific Reports Jun 2017The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to...
The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.
Topics: Alleles; Analysis of Variance; Gene Library; High-Throughput Nucleotide Sequencing; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Sequence Analysis, DNA
PubMed: 28578414
DOI: 10.1038/s41598-017-02727-8 -
Advanced Science (Weinheim,... Nov 2023DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot...
DNA can be used to store digital data, and synthetic short-sequence DNA pools are developed to store high quantities of digital data. However, synthetic DNA data cannot be actively processed in DNA pools. An active DNA data editing process is developed using splint ligation in a droplet-controlled fluidics (DCF) system. DNA fragments of discrete sizes (100-500 bps) are synthesized for droplet assembly, and programmed sequence information exchange occurred. The encoded DNA sequences are processed in series and parallel to synthesize the determined DNA pools, enabling random access using polymerase chain reaction amplification. The sequencing results of the assembled DNA data pools can be orderly aligned for decoding and have high fidelity through address primer scanning. Furthermore, eight 90 bps DNA pools with pixel information (png: 0.27-0.28 kB), encoded by codons, are synthesized to create eight 270 bps DNA pools with an animation movie chip file (mp4: 12 kB) in the DCF system.
Topics: DNA; Polymerase Chain Reaction
PubMed: 37755129
DOI: 10.1002/advs.202303197