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Microbial Biotechnology May 2020The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was...
The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was to explore the feasibility of the ddPCR technique to detect pathogen DNA in whole blood and to assess the diagnostic accuracy of ddPCR to detect bloodstream infections (BSIs), benchmarked against blood cultures. Broad-range primers and probes were designed to detect bacterial 16S rRNA (and Gram stain for differentiation) and fungal 28S rRNA. To determine the detection limit of ddPCR, 10-fold serial dilutions of E. coli and C. albicans were spiked in both PBS and whole blood. The diagnostic accuracy of ddPCR was tested in historically collected frozen blood samples from adult patients suspected of a BSI and compared with blood cultures. Analyses were independently performed by two research analysts. Outcomes included sensitivity and specificity of ddPCR. Within 4 h, blood samples were drawn, and DNA was isolated and analysed. The ddPCR detection limit was approximately 1-2 bacteria or fungi per ddPCR reaction. In total, 45 blood samples were collected from patients, of which 15 (33%) presented with positive blood cultures. The overall sensitivity of ddPCR was 80% (95% CI 52-96) and specificity 87% (95% CI 69-96). In conclusion, the ddPCR technique has considerable potential and is able to detect very low amounts of pathogen DNA in whole blood within 4 h. Currently, ddPCR has a reasonable sensitivity and specificity, but requires further optimization to make it more useful for clinical practice.
Topics: Adult; Candida albicans; DNA Primers; Escherichia coli; Humans; Molecular Diagnostic Techniques; Polymerase Chain Reaction; RNA, Ribosomal, 16S; RNA, Ribosomal, 28S; Sensitivity and Specificity; Sepsis
PubMed: 31605465
DOI: 10.1111/1751-7915.13491 -
Molecules (Basel, Switzerland) Jun 2023This study aimed to explore the applicability of DNA barcoding for assessing the authenticity of caviar on the Chinese market. A set of universal primers and two sets...
This study aimed to explore the applicability of DNA barcoding for assessing the authenticity of caviar on the Chinese market. A set of universal primers and two sets of designed primers based on and genes were used to identify maternal species of samples from 21 batches of caviar. The results showed that the PCR products from three sets of primers had more than 98% similarity to the sequences in database. The gene could not distinguish sturgeons with closed genetic relationships, while gene could effectively improve the accuracy of DNA barcoding and was more suitable to the identification of interspecific sturgeon than the gene. The neighbor-joining dendrogram further confirmed the applicability and accuracy of and genes in identifying maternal relatives of caviar (////. Despite the limitations of mitochondrial DNA in identifying hybrid sturgeon species, the presence of counterfeit caviar of non-sturgeon ingredients could be excluded. All the caviar samples were identified successfully as sturgeon species, but the mislabeling rate of species was 33.4%, indicating that there were illegal phenomena such as disorderly labeling, mislabeling, and adulteration on the market.
Topics: Animals; DNA Barcoding, Taxonomic; DNA, Mitochondrial; Fishes; Polymerase Chain Reaction; DNA Primers
PubMed: 37446706
DOI: 10.3390/molecules28135046 -
Molecular Ecology Resources Jan 2023Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially... (Review)
Review
Dietary metabarcoding has vastly improved our ability to analyse the diets of animals, but it is hampered by a plethora of technical limitations including potentially reduced data output due to the disproportionate amplification of the DNA of the focal predator, here termed "the predator problem". We review the various methods commonly used to overcome this problem, from deeper sequencing to exclusion of predator DNA during PCR, and how they may interfere with increasingly common multipredator-taxon studies. We suggest that multiprimer approaches with an emphasis on achieving both depth and breadth of prey detections may overcome the issue to some extent, although multitaxon studies require further consideration, as highlighted by an empirical example. We also review several alternative methods for reducing the prevalence of predator DNA that are conceptually promising but require additional empirical examination. The predator problem is a key constraint on molecular dietary analyses but, through this synthesis, we hope to guide researchers in overcoming this in an effective and pragmatic way.
Topics: Animals; Food Chain; Predatory Behavior; DNA Primers; Polymerase Chain Reaction; DNA; Diet
PubMed: 36017818
DOI: 10.1111/1755-0998.13705 -
Scientific Reports Nov 2022Cost-effective genotyping can be achieved by sequencing PCR amplicons. Short 3-10 base primers can arbitrarily amplify thousands of loci using only a few primers. To...
Cost-effective genotyping can be achieved by sequencing PCR amplicons. Short 3-10 base primers can arbitrarily amplify thousands of loci using only a few primers. To improve the sequencing efficiency of the multiple arbitrary amplicon sequencing (MAAS) approach, we designed new primers and examined their efficiency in sequencing and genotyping. To demonstrate the effectiveness of our method, we applied it to examining the population structure of the small freshwater fish, medaka (Oryzias latipes). We obtained 2987 informative SNVs with no missing genotype calls for 67 individuals from 15 wild populations and three artificial strains. The estimated phylogenic and population genetic structures of the wild populations were consistent with previous studies, corroborating the accuracy of our genotyping method. We also attempted to reconstruct the genetic backgrounds of a commercial orange mutant strain, Himedaka, which has caused a genetic disturbance in wild populations. Our admixture analysis focusing on Himedaka showed that at least two wild populations had genetically been contributed to the nuclear genome of this mutant strain. Our genotyping methods and results will be useful in quantitative assessments of genetic disturbance by this commercially available strain.
Topics: Animals; Oryzias; Genotyping Techniques; DNA Primers; Genotype; Polymerase Chain Reaction
PubMed: 36411327
DOI: 10.1038/s41598-022-24498-7 -
Talanta Jan 2021Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential...
Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring and vortexing, the only methods that can emulsify libraries of ≥10 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study quantifies the outcome of ePCR from conditions specified in the literature using single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that among the two concentrations of polymerase tested, only the higher one, which is 20-fold more than the concentration recommended for conventional PCR, could yield sufficient PCR products. Dramatic increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.
Topics: DNA Primers; Emulsions; Gene Library; Polymerase Chain Reaction; Single Molecule Imaging
PubMed: 33076127
DOI: 10.1016/j.talanta.2020.121593 -
Nucleic Acids Research Nov 2022DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during...
DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.
Topics: Humans; DNA Polymerase I; Mutagens; DNA Replication; DNA Primase; Mutagenesis; DNA; DNA Primers; RNA
PubMed: 36454017
DOI: 10.1093/nar/gkac1101 -
PloS One 2023The rapid identification of Influenza A virus and its variants, which cause severe respiratory diseases, is imperative to providing timely treatment and improving...
A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan.
The rapid identification of Influenza A virus and its variants, which cause severe respiratory diseases, is imperative to providing timely treatment and improving patient outcomes. Conventionally, two separate assays (total test duration of up to 6 h) are required to initially differentiate Influenza A and B viruses and subsequently distinguish the pdm H1N1 and H3N2 serotypes of Influenza A virus. In this study, we developed a multiplex real-time RT-PCR method for simultaneously detecting Influenza A and B viruses and subtyping Influenza A virus, with a substantially reduced test duration. Clinical specimens from hospitalized patients and outpatients with influenza-like symptoms in Eastern Taiwan were collected between 2011 and 2015, transported to Hualien Tzu Chi Hospital, and analyzed. Conventional RT-PCR was used to subtype the isolated Influenza A viruses. Thereafter, for rapid identification, the multiplex real-time RT-PCR method was developed and applied to identify the conserved regions that aligned with the available primers and probes. Accordingly, a multiplex RT-PCR assay with three groups of primers and probes (MAF and MAR primers and MA probe; InfAF and InfAR primers and InfA probe; and MBF and MBR primers and MB probe) was established to distinguish these viruses in the same reaction. Thus, with this multiplex RT-PCR assay, Influenza B, Influenza A pdm H1N1, and Influenza A H3N2 viruses were accurately detected and differentiated within only 2.5 h. This multiplex RT-PCR assay showed similar analytical sensitivity to the conventional singleplex assay. Further, the phylogenetic analyses of our samples revealed that the characteristics of these viruses were different from those reported previously using samples collected during 2012-2013. In conclusion, we developed a multiplex real-time RT-PCR method for highly efficient and accurate detection and differentiation of Influenza A and B viruses and subtyping Influenza A virus with a substantially reduced test duration for diagnosis.
Topics: Humans; Influenza, Human; Influenza A Virus, H3N2 Subtype; Influenza A Virus, H1N1 Subtype; Taiwan; Phylogeny; Sensitivity and Specificity; Influenza A virus; Mutation; DNA Primers; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 36952488
DOI: 10.1371/journal.pone.0283074 -
Biochemistry Dec 2022and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and...
and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.
Topics: Trypanosoma brucei brucei; DNA, Kinetoplast; DNA Polymerase gamma; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Exonucleases; DNA, Mitochondrial
PubMed: 36399653
DOI: 10.1021/acs.biochem.2c00392 -
Journal of Insect Science (Online) Sep 2023The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from...
The aim of this study was to compare 3 DNA extraction methods: the PureLink Genomic DNA kit, DNAzol Direct reagent, and a microwave-based method, for extracting DNA from an adult Culex quinquefasciatus by focusing on the quantity and purity of DNA, cost, and time required. Ten mosquitoes were individually used for DNA extraction by each method. Based on the results obtained, DNA was extracted from each method using specific primers, resulting in a polymerase chain reaction (PCR) product with a length of 274 bp. The DNA quantity extracted using the DNAzol Direct (179.08 ± 3.77 ng/µl) differs significantly from that of the commercial kit (115.98 ± 4.57 ng/µl) and a microwave-based method (119.26 ± 3.06 ng/µl). The absorbance ratio of DNA extracted using the PureLink Genomic DNA kit, the DNAzol Direct, and the microwave-based methods was 1.92 ± 0.02, 1.79 ± 0.01, and 1.87 ± 0.01, respectively. Among the 3 methods evaluated, the microwave-based method is simpler, less expensive, and more time efficient. This is the first evaluation of the microwave-based method for extracting DNA from an adult mosquito. This study provides a useful guide for alternative DNA extraction methods for PCR-based assays, especially in low-resource settings.
Topics: Animals; Culicidae; Culex; DNA; Polymerase Chain Reaction; DNA Primers
PubMed: 37804500
DOI: 10.1093/jisesa/iead080 -
Nature Communications Jun 2021DNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent...
DNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.
Topics: Computer Simulation; DNA; DNA Primers; Databases, Nucleic Acid; High-Throughput Nucleotide Sequencing; Image Processing, Computer-Assisted; Information Storage and Retrieval; Real-Time Polymerase Chain Reaction; Software; Temperature; Thermodynamics
PubMed: 34112775
DOI: 10.1038/s41467-021-23669-w