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Cold Spring Harbor Protocols Aug 2017Here we describe how mating-type tests are conducted in Two methods can be employed: matings with and tester strains and polymerase chain reaction (PCR) for content.
Here we describe how mating-type tests are conducted in Two methods can be employed: matings with and tester strains and polymerase chain reaction (PCR) for content.
Topics: Genes, Mating Type, Fungal; Genetics, Microbial; Polymerase Chain Reaction; Schizosaccharomyces
PubMed: 28765295
DOI: 10.1101/pdb.prot091728 -
Letters in Applied Microbiology Jun 2023Mycoplasma genitalium is a sexually transmitted infection with increasing concerns around antimicrobial resistance. Droplet digital PCR (ddPCR) is a rapid quantification...
Mycoplasma genitalium is a sexually transmitted infection with increasing concerns around antimicrobial resistance. Droplet digital PCR (ddPCR) is a rapid quantification method with high precision that may be useful for absolute quantitation of bacteria in samples. This study aimed to develop a ddPCR assay for the quantification of M. genitalium. ddPCR targeting the gene mgpB was established and analysed using the QX100 ddPCR system. The assay was evaluated against quantitated DNA standards, and then in comparison to an established quantitative PCR performed on the Lightcycler 480 II. DNA template of increasing complexity was used, including synthetic double stranded DNA, DNA extracts from laboratory-cultured M. genitalium strains (n = 17) and DNA from M. genitalium-positive clinical samples (n = 21). There was a strong correlation between ddPCR concentration estimates and measured DNA standards (r2 = 0.997), and between ddPCR and qPCR quantitation for different templates (r2 ranging from 0.953 to 0.997). ddPCR reliably detected template in a range from <10 copies per reaction to >104 copies per reaction and demonstrated linearity over dilution series. Concentration estimates by ddPCR were reproducibly less than those determine by qPCR. ddPCR demonstrated precise and reproducible quantitation of M. genitalium with a variety of templates.
Topics: Mycoplasma genitalium; Sensitivity and Specificity; Polymerase Chain Reaction; Bacteria; Real-Time Polymerase Chain Reaction
PubMed: 37237449
DOI: 10.1093/lambio/ovad064 -
Molecular Diagnosis & Therapy Apr 2018Digital PCR (dPCR) approaches have been developed for the detection of nucleic acids of low abundance, such as cell-free DNA, and represent an attractive and sensitive... (Review)
Review
Digital PCR (dPCR) approaches have been developed for the detection of nucleic acids of low abundance, such as cell-free DNA, and represent an attractive and sensitive alternative to conventional methods, particularly in the field of non-invasive prenatal diagnosis (NIPD). In this review, we present the principle of dPCR and its applications in the field of prenatal diagnosis from current and emerging uses, such as fetal gender determination, rhesus blood group D antigen genotyping, or monogenic disorders prenatal testing, to future applications, such as the diagnosis and monitoring of pregnancy-related disorders. We also address considerations for implementation of the method in a clinical laboratory and discuss the competiveness of dPCR over other technologies such as quantitative PCR or massively parallel sequencing.
Topics: Fetal Diseases; Humans; Polymerase Chain Reaction; Prenatal Diagnosis
PubMed: 29209991
DOI: 10.1007/s40291-017-0312-x -
Yi Chuan = Hereditas Apr 2020Various derivative technologies based on PCR for nucleic acid detection have emerged with the continuous development and the diverse needs of molecular biology... (Review)
Review
Various derivative technologies based on PCR for nucleic acid detection have emerged with the continuous development and the diverse needs of molecular biology technology. Digital PCR (dPCR) is a nucleic acid detection method for large scale amplification based on a single molecular template, which runs an individual PCR reaction using chambers/wells or droplets. dPCR can be used for absolute quantification for the initial concentration of samples without calibrator and drawing standard curve, showing the characteristics of high sensitivity, specificity, and accuracy. In this review, we introduce the history of technology development, principle, and instrument platform types of digital PCR in detail. Then, we summarize the application of this technology in GMO quantification, disease diagnosis, environment and food supervision. Finally, we describe the application prospect of dPCR, providing a reference for the development and utilization of this technology in the future.
Topics: Polymerase Chain Reaction
PubMed: 32312705
DOI: 10.16288/j.yczz.19-351 -
International Journal of Molecular... May 2019New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on... (Review)
Review
New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods, and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders.
Topics: Biomarkers, Tumor; Humans; Leukemia, Myeloid, Acute; Molecular Diagnostic Techniques; Polymerase Chain Reaction
PubMed: 31067725
DOI: 10.3390/ijms20092249 -
Expert Review of Molecular Diagnostics 2016The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The... (Review)
Review
The remarkable stability of microRNAs in biofluids underlies their potential as biomarkers, but their small size presents challenges for detection by RT-qPCR. The heterogeneity of microRNAs, with each one comprising a series of variants or 'isomiRs', adds additional complexity. Presented here are the key considerations for use of RT-qPCR to measure microRNAs and their isomiRs, with a focus on plasma. Modified nucleotides can be incorporated into primer sequences to enhance affinity and provide increased specificity and sensitivity for RT-qPCR assays. Approaches based upon polyA tailing and use of a common oligo(dT)-based reverse transcription oligonucleotide will detect most isomiRs. Conversely, stem-loop RT oligonucleotides and sequence specific probes can enable detection of specific isomiRs of interest. Next generation sequencing of all the products of a microRNA RT-PCR reaction is a promising new approach for both microRNA quantification and characterization.
Topics: Biomarkers; Humans; MicroRNAs; Molecular Diagnostic Techniques; Polymerase Chain Reaction; Sequence Analysis, RNA
PubMed: 26854938
DOI: 10.1586/14737159.2016.1152184 -
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 -
The Journal of Veterinary Medical... Jun 2016Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective...
Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.
Topics: Animals; Cattle; Enzootic Bovine Leukosis; Leukemia Virus, Bovine; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 26911373
DOI: 10.1292/jvms.15-0577 -
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 -
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