Search results for "Real-time polymerase chain reaction"

Real-Time Polymerase Chain Reaction for Detection of Schistosoma DNA in Small-Volume Urine Samples Reflects Focal Distribution of Urogenital Schistos…

2014

Schistosoma haematobium eggs and Schistosoma DNA levels were measured in urine samples from 708 girls recruited from 18 randomly sampled primary schools in South Africa. Microscopic analysis of two 10-mL urine subsamples collected on three consecutive days confirmed high day-to-day variation; 103 (14.5%) girls had positive results at all six examinations, and at least one positive sample was seen in 225 (31.8%) girls. Schistosoma-specific DNA, which was measured in a 200-μL urine subsample by using real-time polymerase chain reaction, was detected in 180 (25.4%) cases, and levels of DNA corresponded significantly with average urine egg excretion. In concordance with microscopic results, pol…

Bypassing hazard of housekeeping genes: their evaluation in rat granule neurons treated with cerebrospinal fluid of multiple sclerosis subjects

2015

Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS), several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study ai…

A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts

2020

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in‐house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV…

A bacterial metabolite, trimethylamine N-oxide, disrupts the hemostasis balance in human primary endothelial cells but no coagulopathy in mice

2019

: The gut microbial metabolite, trimethylamine N-oxide (TMAO), was previously reported to induce platelet hypersensitivity, which leads to thrombotic risk. However, the molecular mechanism underlying the effects of TMAO on endothelial cells (EC), which is the primary vessel wall contact with the lumen, remains unclear. Here, we investigated the impact of TMAO on procoagulant activity (PCA) in EC and mice, for a possible link between microbiota and coagulation. To test the PCA of TMAO in EC, we performed one-stage clotting assays and converted into PCA. Antitissue factor (TF) antibody was used to test the TF role in PCA. Quantitative PCR was performed to measure the TF, thrombomodulin, IL-6,…

Systemic PaO2 oscillations cause mild brain injury in a pig model

2016

OBJECTIVE: Systemic PaO2 oscillations occur during cyclic recruitment and derecruitment of atelectasis in acute respiratory failure and might harm brain tissue integrity. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult anesthetized pigs. INTERVENTIONS: Pigs were randomized to a control group (anesthesia and extracorporeal circulation for 20 hr with constant PaO2, n = 10) or an oscillation group (anesthesia and extracorporeal circulation for 20 hr with artificial PaO2 oscillations [3 cycles min⁻¹], n = 10). Five additional animals served as native group (n = 5). MEASUREMENTS AND MAIN RESULTS: Outcome following exposure to artificial PaO2 oscillations…

mRNA Induction and Cytokine Release of Inflammatory Mediators During In Vitro Exposure of Human Nasal Respiratory Epithelia to Acetaldehyde

2006

Acetaldehyde has been shown to be cytotoxic and carcinogenic to the upper respiratory tract epithelium of rodents following long-term exposure. Most animal studies have concentrated on carcinogenicity and DNA-protein cross-link formation, while less is known about potential dose- and time-dependent induction of aldehyde-induced rhinitis in humans. In this in vitro study, 22 primary cell cultures established from inferior turbinate tissue of healthy individuals were exposed to acetaldehyde concentrations of 50 (German MAK value) or 500 ppm for 4 or 24 h. mRNA expression and protein levels of cytokines and other inflammatory mediators were quantified at the end of the 4- and 24-h exposures. C…

Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients

2011

Hoerning A, Kalkavan H, Rehme C, Menke J, Worm K, Garritsen HSP, Buscher R, Hoyer PF. Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients. Pediatr Transplantation 2011: 15: 809–818. © 2011 John Wiley & Sons A/S. Abstract:  The presence of microchimerism in peripheral blood of solid organ transplant recipients has been postulated to be beneficial for allograft acceptance. Kinetics of donor cell trafficking and accumulation in pediatric allograft recipients are largely unknown. In this study, we implemented SNPs of the HVRs I and II of mitochondrial DNA to serve as molecular genetic markers to detect donor-specific…

Real-life evaluation of a rapid extraction-free SARS-CoV-2 RT-PCR assay (COVID-19 PCR Fast-L) for the diagnosis of COVID-19.

2021

Comparative evaluation of molecular methods for the quantitative measure of torquetenovirus (TTV) viremia, the new surrogate marker of immune compete…

2019

Background Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict posttransplant immune-related complications, and eventually to customize immunosuppression. Methods In this study, 327 blood samples were tested using two real-time PCR (rtPCR) assays both targeted to the untranslated region of the TTV genome. The first assay was an in-house rtPCR developed by our group, the second one was the recently marketed TTV R-GENE assay. Results In the validation study, the TTV R-GENE showed good performances in precision and reproducibility, and sensitivity as low as 12 TTV DNA copies/mL, like previously reported for the in-house rtPCR. The Bl…

Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using…

2016

International audience; Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a pro…