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The Journal of Veterinary Medical... Aug 2016Canine transmissible venereal tumor (CTVT) is the only naturally contagious tumor that is transmitted during coitus or social behaviors. Based on the tumor's location,...
Canine transmissible venereal tumor (CTVT) is the only naturally contagious tumor that is transmitted during coitus or social behaviors. Based on the tumor's location, the diagnosis of genital TVT (GTVT) is comparably easier than those in the extragenital area (ETVT) that are more easily incorrectly diagnosed. Fortunately, CTVT cells contain a specific long interspersed nuclear elements (LINE), inserted upstream of the myc gene, allowing a diagnostic polymerase chain reaction (PCR) based detection assay. The objectives of this study were aimed to improve the diagnostic accuracy by applying the diagnostic LINE1-c-myc PCR assay and fine needle aspiration (FNA) collection in direct comparison with standard cytological and histopathological analyses. Seventy-four dogs, comprised of 41 and 31 dogs with tumor masses at their external genitalia and extragenital areas (e.g. skin and nasal cavity), respectively, were included in this study. The signalment of these 65 dogs and clinical history of 20 client-owned dogs were collected. Samples were taken by biopsy for both histopathological examination and FNA for cytological examination and diagnostic PCR. The PCR products from 10 apparently CTVT samples were purified and sequenced. Sixty-one CTVT cases were diagnosed by cytological and histological analyses, but 65 were positive by the PCR assay. Overall, the PCR assay improved the accuracy of diagnostic CTVT results, especially for the more difficult ETVT tumors. Moreover, this PCR-based approach can facilitate the decision as to discontinue chemotherapy by discrimination between residual tumor cell masses and fibrotic tissue.
Topics: Animals; DNA, Neoplasm; Dog Diseases; Dogs; Female; Gene Rearrangement; Genes, myc; Long Interspersed Nucleotide Elements; Male; Polymerase Chain Reaction; Venereal Tumors, Veterinary
PubMed: 27075116
DOI: 10.1292/jvms.15-0710 -
Zebrafish Aug 2022Genotyping 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...
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.
Topics: Animals; DNA; Genomics; Polymerase Chain Reaction; Sequence Analysis, DNA; Zebrafish
PubMed: 35877315
DOI: 10.1089/zeb.2022.0015 -
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 -
Analytica Chimica Acta Mar 2016Significant advances have been made in developing microfluidic polymerase chain reaction (PCR) devices in the last two decades. More recently, microfluidic microdroplet... (Review)
Review
Significant advances have been made in developing microfluidic polymerase chain reaction (PCR) devices in the last two decades. More recently, microfluidic microdroplet technology has been exploited to perform PCR in droplets because of its unique features. For example, it can prevent crossover contamination and PCR inhibition, is suitable for single-cell and single-molecule analyses, and has the potential for system integration and automation. This review will therefore focus on recent developments on droplet-based continuous-flow microfluidic PCR, and the major research challenges. This paper will also discuss a new way of on-chip flow control and a rational design simulation tool, which are required to underpin fully integrated and automated droplet-based microfluidic systems. We will conclude with a scientific speculation of future autonomous scientific discoveries enabled by microfluidic microdroplet technologies.
Topics: Automation; Lab-On-A-Chip Devices; Polymerase Chain Reaction; Systems Integration
PubMed: 26965323
DOI: 10.1016/j.aca.2016.02.006 -
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 -
BMC Veterinary Research Jan 2019Canine parvovirus 2 (CPV-2) is one of the most common etiological agents that cause severe gastroenteritis in puppies. Early accurate diagnosis is important for infected...
BACKGROUND
Canine parvovirus 2 (CPV-2) is one of the most common etiological agents that cause severe gastroenteritis in puppies. Early accurate diagnosis is important for infected dogs. In recent years, magnetic separation has become an efficient and useful tool for bioassays. In this study, polymerase chain reaction (PCR) combined with fluorescent lateral flow immunoassay (LFIA) based on magnetic purification assay was developed for the quantitative detection of CPV-2.
RESULTS
The optimum working reaction volume and reaction time for LFIA was 100 μL and 2 min, respectively. The PCR-LFIA assay only detected CPV-2, and did not show cross-detection of non-CPV strains. Experiments showed analytical sensitivity of 3 × 10 copies/μL and demonstrated the PCR-LFIA has a diagnostic agreement of 100% with conventional PCR on detection of clinical samples (22.6% positive, 14/62). Cutoff value is 146. The results were further verified by sequencing and BLAST software. The entire process from PCR step only takes ~ 80 min.
CONCLUSIONS
This approach provides an attractive platform for rapid and quantitative detection of CPV-2, indicating great promise as a convenient molecular detection tool to facilitate disease outbreak investigations and response timely.
Topics: Animals; Dog Diseases; Dogs; Female; Fluoroimmunoassay; Male; Parvoviridae Infections; Parvovirus, Canine; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 30654823
DOI: 10.1186/s12917-019-1774-3 -
Angewandte Chemie (International Ed. in... Jul 2014The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease....
The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3'-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.
Topics: 5-Methylcytosine; HeLa Cells; Humans; Polymerase Chain Reaction
PubMed: 24923910
DOI: 10.1002/anie.201403745 -
BMC Genomics Nov 2021Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) is a sensitive and reliable method for mRNA quantification and rapid analysis of gene expression...
BACKGROUND
Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) is a sensitive and reliable method for mRNA quantification and rapid analysis of gene expression from a large number of starting templates. It is based on the statistical significance of the beginning of exponential phase in real-time PCR kinetics, reflecting quantitative cycle of the initial target quantity and the efficiency of the PCR reaction (the fold increase of product per cycle).
RESULTS
We used the large clinical biomarker dataset and 94-replicates-4-dilutions set which was published previously as research tools, then proposed a new qPCR curve analysis method--CMAN, to determine the position of quantitative cycle as well as the efficiency of the PCR reaction and applied in the calculations. To verify algorithm performance, 20 genes from biomarker and partial data with concentration gradients from 94-replicates-4-dilutions set of MYCN gene were used to compare our method with various publicly available methods and established a suitable evaluation index system.
CONCLUSIONS
The results show that CMAN method is comparable to other methods and can be a feasible method which applied to our self-developed qPCR data processing and analysis software, providing a simple tool for qPCR analysis.
Topics: Gene Expression; Gene Expression Profiling; Genes, myc; Humans; RNA, Messenger; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 34789146
DOI: 10.1186/s12864-021-07986-4 -
The Journal of Investigative Dermatology May 2017In the 3 decades since the discovery of the polymerase chain reaction, a progression of remarkable technical advances has driven great strides in our understanding of... (Review)
Review
In the 3 decades since the discovery of the polymerase chain reaction, a progression of remarkable technical advances has driven great strides in our understanding of molecular biology such that now we are able to study at once the entire and complete set of RNA transcripts that are produced by the genome. In this review, we describe the milestones that have led to this era of global transcriptional analysis, how these approaches have been extended towards skin disease, and their future directions.
Topics: Humans; Molecular Biology; Polymerase Chain Reaction; Skin Diseases; Transcription, Genetic
PubMed: 28411853
DOI: 10.1016/j.jid.2016.02.817 -
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