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Methods in Molecular Biology (Clifton,... 2024Cellular fate is determined by the activity of nuclear transcription factors. Here, we describe a series of protocols for detecting transcription factors at both the...
Cellular fate is determined by the activity of nuclear transcription factors. Here, we describe a series of protocols for detecting transcription factors at both the transcript and protein levels in human adipose cells. Methods for analysis of transcript include RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), endpoint PCR, and RT-qPCR. Evaluation of protein expression includes protocols for protein extraction, quantification by Bradford assay, SDS-PAGE, western blotting, and quantification with ImageJ. Each of these steps is critical for a reliable and reproducible assessment of transcription expression and characterization of human adult-derived adipose stromal/stem cells.
Topics: Adult; Humans; Transcription Factors; Blotting, Western; Reverse Transcriptase Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 38478247
DOI: 10.1007/978-1-0716-3762-3_26 -
Biosensors & Bioelectronics Dec 2023The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens....
The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens. Microfluidics, which involves the manipulation of small volumes of fluidic samples, has been shown to be an ideal approach for POC analysis. Among the various microfluidic platforms available, digital microfluidics (DMF) offers high degree of configurability in manipulating μL/nL-scale liquid and achieving automation. However, the successful implementation of ultrafast PCR on DMF platforms presents challenges due to inherent system instability. In this study, we developed a robust and ultrafast PCR in 3.7-5 min with a detection sensitivity comparable to conventional PCR. Specifically, the implementation of the pincer heating scheme homogenises the temperature within a drop. The utilization of a μm-scale porous hydrophobic membrane suppresses the formation of bubbles under high temperatures. The design of a groove around the high-temperature zone effectively mitigates the temperature interference. The integration of a soluble sensor into the droplets provides an accurate and instant in-drop temperature sensing. We envision that the fast, robust, sensitive, and automatic DMF system will empower the POC testing for infectious diseases.
Topics: Humans; Microfluidics; Biosensing Techniques; Polymerase Chain Reaction; Communicable Diseases; Point-of-Care Systems
PubMed: 37797533
DOI: 10.1016/j.bios.2023.115711 -
Molecular Aspects of Medicine Jun 2024Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time... (Review)
Review
Melting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.
Topics: Nucleic Acid Denaturation; Humans; DNA; Polymerase Chain Reaction
PubMed: 38489863
DOI: 10.1016/j.mam.2024.101268 -
Genes Mar 2024The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The... (Review)
Review
The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.
Topics: Humans; Polymerase Chain Reaction; Forensic Sciences; DNA Fingerprinting; DNA; Forensic Genetics
PubMed: 38674373
DOI: 10.3390/genes15040438 -
Mycopathologia Dec 2023The diagnosis of chronic pulmonary aspergillosis (CPA) is established by combined clinic-radio-microbiological criteria. Out of the different microbiological criteria, a...
The diagnosis of chronic pulmonary aspergillosis (CPA) is established by combined clinic-radio-microbiological criteria. Out of the different microbiological criteria, a positive serology for Aspergillus-specific IgG levels is the cornerstone of diagnosis. Alternatively, other microbiological evidence are sometimes sought viz., positive Aspergillus antigen (broncho-alveolar lavage fluid, i.e., BALF galactomannan ≥ 1.0), histopathological demonstration of the fungi following lung biopsy or resection, demonstration of hyaline septate hyphae in direct microscopy resembling Aspergillus spp. or its growth on a respiratory specimen. However, the exact roles of BALF- GM and the newer BALF-PCR have not been confirmed by studies till date. This study enrolled 210 patients with suspected CPA. Of the participants, 88 patients met the criteria for CPA, whereas 122 patients had an alternative diagnosis. The sensitivity-specificity of AsperGenius® PCR and "in-house" PCR were 52.27(36.69-67.54) %-33.78 (23.19-45.72) % and 36.36 (22.41-52.23) %-39.19 (28.04-51.23) % respectively. The sensitivity/specificity of BALF (> 1.0) and serum galactomannan (> 1.0) were 46.55% (33.34-60.13)/64.08% (54.03-73.3) and 29.82% (22.05-37.6)/86.84% (81.1-92.59) respectively. The optimal cut-off values for BALF-Galactomannan and serum galactomannan in diagnosing CPA were found to be 0.69 (sensitivity: 64%; specificity: 53%) and 0.458 (sensitivity: 67%; specificity: 64%) respectively. This results of this study suggests that Aspergillus PCR from BAL may not be a good "rule-in" test for diagnosing CPA. While the performances of GM in BAL and serum may be better than PCR, it should be best used in conjunction with other clinical, radiological, and other microbiological characteristics.
Topics: Humans; Pulmonary Aspergillosis; Aspergillus; Mannans; Bronchoalveolar Lavage Fluid; Sensitivity and Specificity; Polymerase Chain Reaction; Invasive Pulmonary Aspergillosis
PubMed: 37857979
DOI: 10.1007/s11046-023-00797-z -
The Journal of Applied Laboratory... Jan 2024Digital polymerase chain reaction (dPCR) is an accurate and sensitive molecular method that can be used in clinical diagnostic, prognostic, and predictive tests. The key...
BACKGROUND
Digital polymerase chain reaction (dPCR) is an accurate and sensitive molecular method that can be used in clinical diagnostic, prognostic, and predictive tests. The key component of the dPCR method is the partitioning of a single reaction into many thousands of droplets, nanochannels or other nano- or picoliter-sized reactions. This results in high enough sensitivity to detect rare nucleic acid targets and provides an absolute quantification of target sequences or alleles compared to other PCR-based methods.
CONTENT
An increasing number of dPCR platforms have been introduced commercially in recent years and more are being developed. These platforms differ in the method of partitioning, degree of automation, and multiplexing capabilities but all can be used in similar ways for sensitive and highly accurate quantification of a variety of nucleic acid targets. Currently, clinical applications of dPCR include oncology, microbiology and infectious disease, genetics, and prenatal/newborn screening. Commercially available tests for clinical applications are being developed for variants with diagnostic, prognostic, and therapeutic significance in specific disease types.
SUMMARY
The power of dPCR technology relies on the partitioning of the reactions and results in increased sensitivity and accuracy compared to qPCR. More recently, the sensitivity of dPCR has been applied to the detection of known variants in cell-free DNA and circulating tumor DNA. Future clinical applications of dPCR include liquid biopsy, treatment resistance detection, screening for minimal residual disease, and monitoring allograft engraftment in transplanted patients.
Topics: Pregnancy; Female; Infant, Newborn; Humans; Polymerase Chain Reaction; Prenatal Diagnosis; Nucleic Acids
PubMed: 38167753
DOI: 10.1093/jalm/jfad103 -
Diagnostic Microbiology and Infectious... Jun 2024A 61-year-old male with subacute headache was found to have cryptococcal meningitis despite a negative BioFire FilmArray meningitis/encephalitis panel. This case...
A 61-year-old male with subacute headache was found to have cryptococcal meningitis despite a negative BioFire FilmArray meningitis/encephalitis panel. This case underscores the importance of liberal cryptococcal antigen testing, and that a negative FilmArray panel is inadequate in excluding cryptococcal meningitis, particularly in a HIV-negative host.
Topics: Humans; Meningitis, Cryptococcal; Male; Middle Aged; Polymerase Chain Reaction; Cryptococcus neoformans
PubMed: 38492489
DOI: 10.1016/j.diagmicrobio.2024.116251 -
Scientific Reports Jul 2023The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination...
The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA's integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.
Topics: DNA, Complementary; Reverse Transcription; Polymerase Chain Reaction; DNA; RNA; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 37454173
DOI: 10.1038/s41598-023-38383-4 -
Archives of Razi Institute Aug 2023is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular...
is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of and using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as . The PCR-RFLP assay demonstrated a product for with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for . The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for ; moreover, it is a suitable method for differentiating between and . This technique can detect in a short time with high precision and sensitivity.
Topics: Horses; Animals; Guinea Pigs; Burkholderia mallei; Glanders; Polymorphism, Restriction Fragment Length; Glycerol; Burkholderia pseudomallei; Polymerase Chain Reaction; Horse Diseases
PubMed: 38226390
DOI: 10.32592/ARI.2023.78.4.1305 -
Biosensors & Bioelectronics Feb 2024The limit of detection (LOD), speed, and cost of crucial COVID-19 diagnostic tools, including lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and... (Review)
Review
The limit of detection (LOD), speed, and cost of crucial COVID-19 diagnostic tools, including lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reactions (PCR), have all improved because of the financial and governmental support for the epidemic. The most notable improvement in overall efficiency among them has been seen with PCR. Its significance for human health increased during the COVID-19 pandemic, when it emerged as the commonly used approach for identifying the virus. However, because of problems with speed, complexity, and expense, PCR deployment in point-of-care settings continues to be difficult. Microfluidic platforms offer a promising solution by enabling the development of smaller, more affordable, and faster PCR systems. In this review, we delve into the engineering challenges associated with the advancement of high-speed microfluidic PCR equipment. We introduce criteria that facilitate the evaluation and comparison of factors such as speed, LOD, cycling efficiency, and multiplexing capacity, considering sample volume, fluidics, PCR reactor geometry and materials, as well as heating/cooling methods. We also provide a comprehensive list of commercially available PCR devices and conclude with projections and a discussion regarding the current obstacles that need to be addressed in order to progress further in this field.
Topics: Humans; COVID-19; Pandemics; Biosensing Techniques; Polymerase Chain Reaction; Microfluidics; COVID-19 Testing
PubMed: 38039729
DOI: 10.1016/j.bios.2023.115830