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Journal of Visualized Experiments : JoVE Sep 2018Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands...
Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.
Topics: Circulating Tumor DNA; Humans; Mutation; Polymerase Chain Reaction
PubMed: 30320738
DOI: 10.3791/58051 -
Journal of Fish Diseases Sep 2023Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase...
Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.
Topics: Animals; Tilapia; Reproducibility of Results; Fish Diseases; Polymerase Chain Reaction; Viruses; Real-Time Polymerase Chain Reaction
PubMed: 37294665
DOI: 10.1111/jfd.13816 -
Cold Spring Harbor Protocols Sep 2021This protocol describes the use of TOPO-activated TA vectors for cloning. Manufacturers of cloning kits provide excellent manuals that explain in detail what to do and...
This protocol describes the use of TOPO-activated TA vectors for cloning. Manufacturers of cloning kits provide excellent manuals that explain in detail what to do and why to do it. This makes TOPO cloning easy, but not foolproof. When setting up TOPO cloning for the first time, set up a trial experiment as described here.
Topics: Cloning, Molecular; Genetic Vectors; Polymerase Chain Reaction
PubMed: 34470863
DOI: 10.1101/pdb.prot101311 -
Analytical Chemistry Mar 2022Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including...
Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh-Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/μL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.
Topics: DNA; Microfluidic Analytical Techniques; Microfluidics; Nucleic Acids; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 35200004
DOI: 10.1021/acs.analchem.1c04983 -
The Veterinary Record Aug 2021
Topics: Animals; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 34415606
DOI: 10.1002/vetr.853 -
International Journal of... 2022In this study, it was aimed to investigate Mycobacterium bovis strains isolated from lungs and lymph nodes of slaughtered animals on clonal level by using different...
Spoligotyping and polymerase chain reaction based strains typing with methods (enterobacterial repetitive intergenic consensus-polymerase chain reaction, randomly amplified polymorphic dnas-polymerase chain reaction and out polymerase chain reaction).
BACKGROUND
In this study, it was aimed to investigate Mycobacterium bovis strains isolated from lungs and lymph nodes of slaughtered animals on clonal level by using different methods such as spoligotyping, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNAs (RAPD-PCR) and OUT-PCR. Comparative evaluation of these methods was further conducted.
METHODS
A total of 38 M. bovis isolates were evaluated in the study. DNA isolation of all M. bovis strains isolated from pruvat free Löwenstein Jensen medium was done by boiling method for ERIC-PCR, RAPD-PCR, and OUT PCR. Mickle device was used for DNA isolation for spoligotyping method.
RESULTS
In 38 M. bovis isolates examined in our study, 4 different groups were determined by spoligotyping and RAPD-PCR test methods, and 5 different groups were detected in ERIC-PCR tests. In the OUT-PCR tests, the band which provides sufficient type separation was not observed.
CONCLUSION
ERIC-PCR, RAPD-PCR, and OUT-PCR methods are easily applicable, simple, and relatively inexpensive methods for evaluating the differences between origins in the typing of M. bovis. The tests need to be evaluated in more detail with extensive studies.
Topics: Animals; Bacterial Typing Techniques; Consensus; DNA; DNA, Bacterial; Enterobacteriaceae; Humans; Mycobacterium bovis; Polymerase Chain Reaction; Random Amplified Polymorphic DNA Technique
PubMed: 35295029
DOI: 10.4103/ijmy.ijmy_253_21 -
Analytical Chemistry Jul 2022Here, a fluorescent polymerase chain reaction (PCR) nanokit is established to detect the specific DNA sequence in a single living cell. Different from well-developed...
Here, a fluorescent polymerase chain reaction (PCR) nanokit is established to detect the specific DNA sequence in a single living cell. Different from well-developed protocols to load cell-permeable probes into single cell for recognition, the DNA sequence in a cellular nucleus is sorted into a nanopipette in our strategy. The target DNA sequence is reacted with the PCR kit components in the nanopipette to complete a PCR amplification reaction. SYBR Green prefilled in the nanopipette is intercalated into double-stranded DNA to induce fluorescence emission for real-time detection down to a single copy. An obvious increase in the fluorescence is observed that validates the detection of the target DNA sequence in single living cells. The established real-time fluorescent PCR nanokit could adapt the PCR kit for single cell analysis and thus offers an alternatively general and highly sensitive strategy for the detection of specific DNA sequences in single living cells.
Topics: Base Sequence; DNA; Fluorescent Dyes; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Single-Cell Analysis
PubMed: 35833720
DOI: 10.1021/acs.analchem.2c02470 -
PloS One 2015The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of...
The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3-12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled "Polymerase-exonuclease (PEX) PCR", in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.
Topics: Base Pair Mismatch; DNA Primers; Polymerase Chain Reaction
PubMed: 25996930
DOI: 10.1371/journal.pone.0128122 -
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 -
Animal Health Research Reviews Dec 2022In basic research, testing of oral fluid specimens by real-time quantitative polymerase chain reaction (qPCR) has been used to evaluate changes in gene expression levels... (Review)
Review
In basic research, testing of oral fluid specimens by real-time quantitative polymerase chain reaction (qPCR) has been used to evaluate changes in gene expression levels following experimental treatments. In diagnostic medicine, qPCR has been used to detect DNA/RNA transcripts indicative of bacterial or viral infections. Normalization of qPCR using endogenous and exogenous reference genes is a well-established strategy for ensuring result comparability by controlling sample-to-sample variation introduced during sampling, storage, and qPCR testing. In this review, the majority of recent publications in human ( = 136) and veterinary ( = 179) medicine did not describe the use of internal reference genes in qPCRs for oral fluid specimens (52.9% animal studies; 57.0% human studies). However, the use of endogenous reference genes has not been fully explored or validated for oral fluid specimens. The lack of valid internal reference genes inherent to the oral fluid matrix will continue to hamper the reliability, reproducibility, and generalizability of oral fluid qPCR assays until this issue is addressed.
Topics: Humans; Animals; Reproducibility of Results; Real-Time Polymerase Chain Reaction
PubMed: 36330795
DOI: 10.1017/S1466252322000044