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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 -
Molecular and Cellular Biochemistry Jul 2023Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore... (Review)
Review
Gene mutation has been a concern for researchers because it results in genetic variations with base changes in molecular structure. Researchers continue to explore methods to detect gene mutations, which may help in disease diagnosis, medication guidance, and so on. Currently, the detection methods, such as whole-genome sequencing and polymerase chain reaction, have some limitations in terms of cost and sensitivity. Ligase (an enzyme) can recognize base mismatch as a commonly used tool in genetic engineering. Therefore, the ligase-related nucleic acid amplification technology for detecting gene mutations has become a research hotspot. In this study, the main techniques explored for detecting gene mutations included the ligase detection reaction, ligase chain reaction, rolling circle amplification reaction, enzyme-assisted polymerase chain reaction, and loop-mediated isothermal amplification reaction. This review aimed to analyze the aforementioned techniques and mainly present their advantages and disadvantages, sensitivity, specificity, cost, detection time, applications, and so on. The findings may help develop sufficient grounds for further studies on detecting gene mutations.
Topics: Ligases; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Mutation; Technology; Nucleic Acids
PubMed: 36441353
DOI: 10.1007/s11010-022-04615-w -
PLoS Medicine Nov 2023Rapid and accurate detection of pathogens is needed in community-acquired pneumonia (CAP) to enable appropriate antibiotics and to slow the development of antibiotic... (Randomized Controlled Trial)
Randomized Controlled Trial
Evaluation of point-of-care multiplex polymerase chain reaction in guiding antibiotic treatment of patients acutely admitted with suspected community-acquired pneumonia in Denmark: A multicentre randomised controlled trial.
BACKGROUND
Rapid and accurate detection of pathogens is needed in community-acquired pneumonia (CAP) to enable appropriate antibiotics and to slow the development of antibiotic resistance. We aimed to compare the effect of point-of-care (POC) polymerase chain reaction (PCR) detection of respiratory pathogens added to standard care with standard care only (SCO) on antibiotic prescriptions after acute hospital admission.
METHODS AND FINDINGS
We performed a superiority, parallel-group, open-label, multicentre, randomised controlled trial (RCT) in 3 Danish medical emergency departments (EDs) from March 2021 to February 2022. Adults acutely admitted with suspected CAP during the daytime on weekdays were included and randomly assigned (1:1) to POC-PCR (The Biofire FilmArray Pneumonia Panel plus added to standard care) or SCO (routine culture and, if requested by the attending physician, target-specific PCR) analysis of respiratory samples. We randomly assigned 294 patients with successfully collected samples (tracheal secretion 78.4% or expectorated sputum 21.6%) to POC-PCR (n = 148, 50.4%) or SCO (146, 49.6%). Patients and investigators owning the data were blinded to the allocation and test results. Outcome adjudicators and clinical staff at the ED were not blinded to allocation and test results but were together with the statistician, blinded to data management and analysis. Laboratory staff performing standard care analyses was blinded to allocation. The study coordinator was not blinded. Intention-to-treat and per protocol analysis were performed using logistic regression with Huber-White clustered standard errors for the prescription of antibiotic treatment. Loss to follow-up comprises 3 patients in the POC-PCR (2%) and none in the SCO group. Intention-to-treat analysis showed no difference in the primary outcome of prescriptions of no or narrow-spectrum antibiotics at 4 h after admission for the POC-PCR (n = 91, 62.8%) odds ratio (OR) 1.13; (95% confidence interval (CI) [0.96, 1.34] p = 0.134) and SCO (n = 87, 59.6%). Secondary outcomes showed that prescriptions were significantly more targeted at 4-h OR 5.68; (95% CI [2.49, 12.94] p < 0.001) and 48-h OR 4.20; (95% CI [1.87, 9.40] p < 0.001) and more adequate at 48-h OR 2.11; (95% CI [1.23, 3.61] p = 0.006) and on day 5 in the POC-PCR group OR 1.40; (95% CI [1.18, 1.66] p < 0.001). There was no difference between the groups in relation to intensive care unit (ICU) admissions OR 0.54; (95% CI [0.10, 2.91] p = 0.475), readmission within 30 days OR 0.90; (95% CI [0.43, 1.86] p = 0.787), length of stay (LOS) IRR 0.82; (95% CI [0.63, 1.07] p = 0.164), 30 days mortality OR 1.24; (95% CI [0.32, 4.82] p = 0.749), and in-hospital mortality OR 0.98; (95% CI [0.19, 5.06] p = 0.986).
CONCLUSIONS
In a setting with an already restrictive use of antibiotics, adding POC-PCR to the diagnostic setup did not increase the number of patients treated with narrow-spectrum or without antibiotics. POC-PCR may result in a more targeted and adequate use of antibiotics. A significant study limitation was the concurrent Coronavirus Disease 2019 (COVID-19) pandemic resulting in an unusually low transmission of respiratory virus.
TRIAL REGISTRATION
ClinicalTrials.gov (NCT04651712).
Topics: Adult; Humans; Point-of-Care Systems; Multiplex Polymerase Chain Reaction; COVID-19; Anti-Bacterial Agents; Denmark; COVID-19 Testing
PubMed: 38015833
DOI: 10.1371/journal.pmed.1004314 -
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 -
Graefe's Archive For Clinical and... Jul 2023Acanthamoeba keratitis (AK) is a painful and possibly sight-threatening ocular infection. While the correct diagnosis and specific treatment in the early stages...
PURPOSE
Acanthamoeba keratitis (AK) is a painful and possibly sight-threatening ocular infection. While the correct diagnosis and specific treatment in the early stages significantly improve the prognosis, the disease is often misdiagnosed and in clinical examination confused with other forms of keratitis. Polymerase chain reaction (PCR) for the detection of AK was first introduced in our institution in December 2013 to improve the timely diagnosis of AK. The aim of this study was to assess the impact of implementation of Acanthamoeba PCR on the diagnosis and treatment of the disease in a German tertiary referral center.
PATIENTS AND METHODS
Patients treated for Acanthamoeba keratitis between 1st of January 1993 and 31st of December 2021 in the Department of Ophthalmology of the University Hospital Duesseldorf were identified retrospectively via in-house registries. Evaluated parameters include age, sex, initial diagnosis, method of correct diagnosis, duration of symptoms until correct diagnosis, contact lens use, visual acuity, and clinical findings as well as medical and surgical therapy by keratoplasty (pKP). In order to assess the impact of implementation of Acanthamoeba PCR, the cases were divided into two groups (before (pre-PCR group) and after PCR implementation (PCR group).
RESULTS
Seventy-five patients with Acanthamoeba keratitis were included (69.3% female, median age 37 years). Eighty-four percent (63/75) of all patients were contact lens wearers. Until PCR was available, 58 patients with Acanthamoeba keratitis were diagnosed either clinically (n = 28), by histology (n = 21), culture (n = 6), or confocal microscopy (n = 2) with a median duration until diagnosis of 68 (18; 109) days. After PCR implementation, in 17 patients, the diagnosis was established with PCR in 94% (n = 16) and median duration until diagnosis was significantly shorter with 15 (10; 30.5) days. A longer duration until correct diagnosis correlated with a worse initial visual acuity (p = 0.0019, r = 0.363). The number of pKP performed was significantly lower in the PCR group (5/17; 29.4%) than in the pre-PCR group (35/58; 60.3%) (p = 0.025).
CONCLUSIONS
The choice of diagnostic method and especially the application of PCR have a significant impact on the time to diagnosis and on the clinical findings at the time of confirmation of diagnosis and the need for penetrating keratoplasty. In contact lens-associated keratitis, the first crucial step is to take AK into consideration and perform a PCR test as timely confirmation of diagnosis of AK is imperative to prevent long-term ocular morbidity.
Topics: Humans; Female; Adult; Male; Acanthamoeba Keratitis; Retrospective Studies; Acanthamoeba; Polymerase Chain Reaction; Disease Progression
PubMed: 36795161
DOI: 10.1007/s00417-023-05993-7 -
International Journal of Molecular... Jul 2023DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an...
DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing DNA polymerase for routine use in laboratories. We developed a method using () that expresses thermostable DNA polymerase directly in the PCR without purification. The gene was transformed into and expressed. After overnight incubation and washing, -expressing DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.
Topics: Taq Polymerase; Escherichia coli; Polymerase Chain Reaction; DNA; DNA Replication
PubMed: 37511160
DOI: 10.3390/ijms241411405 -
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 -
Talanta Aug 2023Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain... (Review)
Review
Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.
Topics: Humans; COVID-19; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; DNA-Directed DNA Polymerase; Nucleic Acids
PubMed: 37148686
DOI: 10.1016/j.talanta.2023.124645 -
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 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y