Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A Rapid and Noninvasive Method That Extracts Polymerase Chain Reaction-Ready Genomic DNA from Adult Zebrafish.

ABSTRACT: Genotyping usually entails analysis of the products of polymerase chain reaction (PCR) carried out with genomic DNA (gDNA) as template, and is employed for validation of mutant or transgenic organisms. For genotyping of adult zebrafish, gDNA is often extracted from clipped caudal fin or skin mucus through either alkaline lysis using NaOH or proteinase K (PK) treatment. Further purification of the gDNA using ethanol precipitation was optional. To develop a rapid and noninvasive method that extracts PCR-ready gDNA from adult zebrafish, we combined skin swabbing with PK treatment and demonstrated its efficiency. This method could be applied to a wide range of fish.

SUBMITTER: Jang JY

PROVIDER: S-EPMC9419989 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A Rapid and Noninvasive Method That Extracts Polymerase Chain Reaction-Ready Genomic DNA from Adult Zebrafish.

Jang Ji Yun JY Liang Ting T Kim Myeong-Kyu MK Kang Kyung Wook KW Lee Bora B Choi Seok-Yong SY

Zebrafish 20220726 4

Genotyping usually entails analysis of the products of polymerase chain reaction (PCR) carried out with genomic DNA (gDNA) as template, and is employed for validation of mutant or transgenic organisms. For genotyping of adult zebrafish, gDNA is often extracted from clipped caudal fin or skin mucus through either alkaline lysis using NaOH or proteinase K (PK) treatment. Further purification of the gDNA using ethanol precipitation was optional. To develop a rapid and noninvasive method that extrac ...[more]

PMID: 35877315

Similar Datasets

A rapid identification method for common astigmatid species based on multiplex polymerase chain reaction.

Project description:Astigmatid mites are economically significant pests of stored products and sources of inhalant allergens causing allergic rhinitis and asthma worldwide. The morphological identification of astigmatid mites at the species level is often a difficult task due to their small size, phenotypic similarity and lack of diagnostic characters. We used multiplex polymerase chain reaction (PCR) to identify astigmatid mite species, which could complement the morphological data for the species-specific identification of mites. Internal ribosomal transcribed spacer (ITS) sequences (i.e., partial 18S, the full length of ITS1-5.8S-ITS2 and partial 28S) from eight astigmatid species (Acarus siro, Tyrophagus putrescentiae, Suidasia nesbitti, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Lepidoglyphus destructor, Chortoglyphus arcuatus and Gohieria fuscus) were obtained by DNA extraction and then sequenced after PCR amplification. Specific primers were designed in the ITS2 region manually. Results revealed that an identification method for eight common astigmatid species was established based on multiplex PCR, which should be effective for the identification of other species of mites by redesigning species-specific primers in future experiments.

| S-EPMC9840793 | biostudies-literature

A specific polymerase chain reaction method to identify Stenotrophomonas maltophilia.

Project description:Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that is difficult to identify unequivocally using current methods. Accordingly, because the presence of this microorganism in a patient may directly determine the antimicrobial treatment, conventional polymerase chain reaction (PCR) and real-time PCR assays targeting 23S rRNA were developed for the specific identification of S. maltophilia. The PCR protocol showed high specificity when tested against other species of Stenotrophomonas, non-fermentative Gram-negative bacilli and 100 clinical isolates of S. maltophilia previously identified using the Vitek system.

| S-EPMC4005581 | biostudies-literature

Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer.

Project description:Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

| S-EPMC9002326 | biostudies-literature

Droplet digital polymerase chain reaction for rapid broad-spectrum detection of bloodstream infections.

Project description: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.

| S-EPMC7111091 | biostudies-literature

Microdevice-based solid-phase polymerase chain reaction for rapid detection of pathogenic microorganisms.

Project description: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.

| S-EPMC7161830 | biostudies-literature

Mirror-image polymerase chain reaction.

Project description:The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp l-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction, and that both the natural and mirror-image systems operate with strict chiral specificity. The development of efficient miPCR systems may lead to many practical applications, such as mirror-image systematic evolution of ligands by exponential enrichment for the selection of therapeutically promising nuclease-resistant l-nucleic acid aptamers.

| S-EPMC5643884 | biostudies-literature

Using real-time polymerase chain reaction as an alternative rapid method for enumeration of colony count in live <i>Brucella</i> vaccines.

Project description:AimBrucellosis is a major bacterial zoonosis of global importance affecting a range of animal species and man worldwide. It has economic, public health, and bio-risk importance. Control and prevention of animal brucellosis mainly depend on accurate diagnostic tools and implementation of effective and safe animal vaccination program. There are three types of animal Brucella live vaccines - Brucella melitensis Rev-1 vaccine, Brucella abortus S19, and B. abortus RB51. Evaluation of these vaccines depends mainly on enumeration of Brucella viable count. At present, used colony count method is time consuming, costly and requires especial skills. Hence, the aim of this study is to use and standardize real-time polymerase chain reaction (RT-PCR) as an alternative, quantitative, sensitive, and rapid method to detect the colony count of Brucella in live Brucella vaccine.Materials and methodsFour batches of different live Brucella vaccines were evaluated using of conventional bacterial count and RT-quantitative PCR (RT-qPCR) using BSCP31 gene specific primers and probe. Standard curve was generated from DNA template extracted from 10-fold serial dilution of living B. abortus RB51 vaccine to evaluate the sensitivity of RT-qPCR.ResultsResults revealed that three batches of living Brucella vaccines were acceptable for Brucella colony count when traditional bacterial enumeration method was used. Results of RT-qPCR were identical to that of conventional bacterial count.ConclusionsResults concluded that RT-qPCR was relatively sensitive compared to traditional bacterial colony count of these vaccines.

| S-EPMC5499076 | biostudies-literature

Application of a reliable and rapid polymerase chain reaction based method in the diagnosis of myotonic dystrophy type 1 (DM1) in India.

Project description:DM1 is caused by CTG repeat expansion in the 3'-UTR of the DMPK gene. DM1 patients have expansions of greater than 50 repeats and up to many thousands. The intention of the present study is the establishment of reliable and rapid polymerase chain reaction methodology in early screening of DM1 patients and their family members. PCR followed by TP-PCR was assessed for screening of 27 cases (from 26 families) and 75 family members and 300 control samples. All patients had CTG repeat expansion while forty seven (63%) and twenty eight (37%), out of seventy five family members were heterozygous and homozygous respectively. Similarly, two hundred thirty (76.77%) and seventy (23.33%), out of three hundred control subjects were heterozygous and homozygous respectively and the number of repeats varied from 5 to 35. Thirteen complete family screenings were done. Thus, TP-PCR is a reliable and rapid molecular technique for the detection of CTG repeat expansion in DM1.

| S-EPMC4287798 | biostudies-literature

Target-selective joint polymerase chain reaction: a robust and rapid method for high-throughput production of recombinant monoclonal antibodies from single cells.

Project description:BackgroundDuring the development of a therapeutic antibody, large numbers of monoclonal antibodies are required to screen for those that are best suited for the desired activity. Although the single cell-based immunoglobulin variable gene cloning technique is a powerful tool, the current methods remain an obstacle to the rapid production of large numbers of recombinant antibodies.ResultsWe have developed a novel overlap extension polymerase chain reaction, the target-selective joint polymerase chain reaction (TS-jPCR), and applied it to the generation of linear immunoglobulin gene expression constructs. TS-jPCR is conducted using a PCR-amplified immunoglobulin variable gene and an immunoglobulin gene-selective cassette (Ig-cassette) that contains all essential elements for antibody expression and overlapping areas of immunoglobulin gene-specific homology. The TS-jPCR technique is simple and specific; the 3'-random nucleotide-tailed immunoglobulin variable gene fragment and the Ig-cassette are assembled into a linear immunoglobulin expression construct, even in the presence of nonspecifically amplified DNA. We also developed a robotic magnetic beads handling instrument for single cell-based cDNA synthesis to amplify immunoglobulin variable genes by rapid amplification of 5' cDNA ends PCR. Using these methods, we were able to produce recombinant monoclonal antibodies from large numbers of single plasma cells within four days.ConclusionOur system reduces the burden of antibody discovery and engineering by rapidly producing large numbers of recombinant monoclonal antibodies in a short period of time.

| S-EPMC3155496 | biostudies-literature

Detecting Malaria Hotspots: A Comparison of Rapid Diagnostic Test, Microscopy, and Polymerase Chain Reaction.

Project description:BackgroundMalaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used.MethodsWe conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic.ResultsEight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT.ConclusionMalaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls.

| S-EPMC5853881 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data