Search results for "Serial dilution"
showing 10 items of 32 documents
Quantification of Listeria monocytogenes in salads by real time quantitative PCR
2005
Abstract A real time quantitative PCR (RTQ-PCR) was carried out purifying DNA extracts of Listeria monocytogenes using a High Pure Listeria Sample Preparation Kit and quantifying in a LightCycler system with hybridisation probes. A standard curve was constructed with serial dilutions. A range linear relationship, from 10 to 10 5 L. monocytogenes colony forming units (CFU), was observed between threshold cycle ( C t ) and logarithmic concentration of the serial dilutions. The assay was linear in a range from 10 to 10 5 L. monocytogenes CFU and the coefficient of determination ( r 2 ) was > 0.98. RTQ-PCR presented an efficiency of > 85%. The accuracy of the PCR-based assay, expressed as % bia…
Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples
2009
A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of …
A TaqMan-based real-time PCR assay for the specific detection and quantification ofLeuconostoc mesenteroidesin meat products
2007
A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified …
Evaluation of a Real-Time PCR Assay for the Detection and Quantification of Group Spores in Food
2010
A procedure based on quantitative real-time PCR was evaluated for the detection and quantification of Bacillus cereus spores. Several methods for DNA isolation, such as various heat treatments and germination solutions, were evaluated on spore suspensions of representative strains of the B. cereus group. Overall, the commercially available DNeasy tissue kit yielded the maximum amount of DNA. The procedure also was used to construct calibration curves for different food matrices, with a wide spore quantification range of 5 log units using serial dilutions of spore suspensions of B. cereus CECT 148T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted i…
Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
2016
Real-time reverse transcription polymerase chain reaction (PCR) is the gold standard for expression analysis. Designed to improve reproducibility and sensitivity, commercial kits are commonly used for the critical step of cDNA synthesis. The present study was designed to determine the impact of these kits. mRNA from mouse brains were pooled to create serial dilutions ranging from 0.0625 μg to 2 μg, which were transcribed into cDNA using four different commercial reverse-transcription kits. Next, we transcribed mRNA from brain tissue after acute brain injury and naïve mice into cDNA for qPCR. Depending on tested genes, some kits failed to show linear results in dilution series and revealed s…
Concordance of assays designed for the quantification of JAK2V617F: a multicenter study
2008
Background Many different techniques have been designed for the quantification of JAK2 V617F allelic burden, sometimes producing discrepant results. Design and Methods JAK2 V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. Results A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniq…
Development and validation of a radioimmunoassay for serum melatonin.
1989
A radioimmunoassay using N-[3-(4-hydroxy-3-[4-hydroxy-3- [125I]iodophenylpropionyl)]-5-methoxytryptamine as tracer for determination of melatonin in the serum of different species is described. Melatonin antisera were raised in rabbits by immunization with a bovine serum albumin conjugate of N-[3-(2-aminoethyl)-5-methoxy indole] hemisuccinamide. A single high affinity, specific antiserum was obtained. In contrast to previous studies, the tracer was synthesised in one step in the absence of water, giving an excellent yield of highly pure product. No chromatographic purification step was needed. Polyethylene glycol in combination with goat antirabbit immunoglobulins was used to separate bound…
The role of antibiotic prophylaxis in reducing bacterial contamination of autologous bone graft collected from implant site
2017
The aim of this study was to evaluate if antibiotic prophylaxis reduces the bacterial contamination of bone particles collected directly from the burs used for implant site preparation. Thirty-four patients underwent the surgical procedures for a total of 34 implant sites. One 1 gr. tablet of amoxicillin + clavulanic acid was given to the test group 12 hours and 1 hour before the surgery. The control group did not take antibiotic prophylaxis. Bone particles were collected and centrifuged. The suspensions were subjected to serial dilutions and each dilution was examined twice using a spatulation technique in Trypticase Soy Agar (TSA), in Sabouraud Dextrose Agar, and in Mitis Salivarius Agar …
Comparison of iodinated contrast media for the assessment of atherosclerotic plaque attenuation values by CT coronary angiography: observations in an…
2013
Objective: To compare the influence of different iodinated contrast media with several dilutions on plaque attenuation in an ex vivo coronary model studied by multislice CT coronary angiography. Methods: In six ex vivo left anterior descending coronary arteries immersed in oil, CT (slices/collimation 64x0.625 mm, temporal resolution 210 ms, pitch 0.2) was performed after intracoronary injection of a saline solution, and solutions of a dimeric isosmolar contrast medium (Iodixanol 320 mgl ml(-1)) and a monomeric high-iodinated contrast medium (Iomeprol 400 mgl ml(-1)) with dilutions of 1/80 (low concentration), 1/50 (medium concentration), 1/40 (high concentration) and 1/20 (very high concent…
Construction of simplified microbial consortia to degrade recalcitrant materials based on enrichment and dilution-to-extinction cultures
2019
AbstractThe capacity of microbes degrading recalcitrant materials has been extensively explored from environmental remediation to industrial applications. Although significant achievements were obtained with single strains, focus is now going toward the use of microbial consortia because of advantages in terms of functional stability and efficiency. While consortia assembly attempts were made from several known single strains, another approach consists in obtaining consortia from complex environmental microbial communities in search for novel microbial species, genes and functions. However, assembling efficient microbial consortia from complex environmental communities is far from trivial d…