Optimization of quantitative polymerase chain reactions for detection and quantification of eight periodontal bacterial pathogens

BACKGROUND: The aim of this study was to optimize quantitative (real-time) polymerase chain reaction (qPCR) assays for 8 major periodontal pathogens, i.e. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micros, Porphyromonas gingivalis, Prevotella intermedia, Tanerella forsythia and Treponema denticola, and of the caries pathogen Streptococcus mutans. RESULTS: Eighteen different primer pairs were analyzed in silico regarding specificity (using BLAST analysis) and the presence of secondary structures at primer binding sites (using mFOLD). The most specific and efficiently binding primer pairs, according to these analyses, were selected for qPCR-analysis to determine amplification efficiency, limit of quantification and intra-run reproducibility. For the selected primer pairs, one for each species, the specificity was confirmed by assessing amplification of DNA extracts from isolates of closely related species. For these primer pairs, the intercycler portability was evaluated on 3 different thermal cyclers (the Applied Biosystems 7300, the Bio-Rad iQ5 and the Roche Light Cycler 480). For all assays on the different cyclers, a good correlation of the standard series was obtained (i.e. r2 >= 0.98), but quantification limits varied among cyclers. The overall best quantification limit was obtained by using a 2 mul sample in a final volume of 10 mul on the Light Cycler 480. CONCLUSIONS: In conclusion, the proposed assays allow to quantify the bacterial loads of S. mutans, 6 periodontal pathogenic species and the genus Fusobacterium.This can be of use in assessing periodontal risk, determination of the optimal periodontal therapy and evaluation of this treatment

Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women

Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS.status: publishe

Microflora of the penile skin-lined neovagina of transsexual women

<p>Abstract</p> <p>Background</p> <p>The microflora of the penile skin-lined neovagina in male-to-female transsexuals is a recently created microbial niche which thus far has been characterized only to a very limited extent. Yet the knowledge of this microflora can be considered as essential to the follow-up of transsexual women. The primary objective of this study was to map the neo-vaginal microflora in a group of 50 transsexual women for whom a neovagina was constructed by means of the inverted penile skin flap technique. Secondary objectives were to describe possible correlations of this microflora with multiple patients' characteristics, such as sexual orientation, the incidence of vaginal irritation and malodorous vaginal discharge.</p> <p>Results</p> <p>Based on Gram stain the majority of smears revealed a mixed microflora that had some similarity with bacterial vaginosis (BV) microflora and that contained various amounts of cocci, polymorphous Gram-negative and Gram-positive rods, often with fusiform and comma-shaped rods, and sometimes even with spirochetes. <it>Candida </it>cells were not seen in any of the smears.</p> <p>On average 8.6 species were cultured per woman. The species most often found were: <it>Staphylococcus epidermidis</it>, <it>Streptococcus anginosus </it>group spp., <it>Enterococcus faecalis, Corynebacterium </it>sp., <it>Mobiluncus curtisii </it>and <it>Bacteroides ureolyticus</it>. Lactobacilli were found in only one of 30 women</p> <p>There was no correlation between dilatation habits, having coitus, rinsing habits and malodorous vaginal discharge on the one hand and the presence of a particular species on the other. There was however a highly significant correlation between the presence of <it>E. faecalis </it>on the one hand and sexual orientation and coitus on the other (p = 0.003 and p = 0.027 respectively).</p> <p>Respectively 82%, 58% and 30% of the samples showed an amplicon after amplification with <it>M. curtisii</it>, <it>Atopobium vaginae </it>and <it>Gardnerella vaginalis </it>primer sets.</p> <p>Conclusion</p> <p>Our study is the first to describe the microflora of the penile skin-lined neovagina of transsexual women. It reveals a mixed microflora of aerobe and anaerobe species usually found either on the skin, in the intestinal microflora or in a BV microflora.</p

The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniae and comparison with four other species specific PCR assays

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge.</p> <p>Methods</p> <p>This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of <it>S. pneumoniae</it>, and its performance compared to other genotypic and phenotypic tests.</p> <p>Results</p> <p>The new PCR assay designed in this study, proved to be specific at 57°C for <it>S. pneumoniae</it>, not amplifying <it>S. pseudopneumoniae </it>or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays (psaA, LytA, ply, spn9802-PCR) were previously described for the specific amplification of <it>S. pneumoniae</it>, but <it>psaA</it>-PCR was the only one found not to cross-react with <it>S. pseudopneumoniae</it>.</p> <p>Conclusion</p> <p>Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify <it>S. pseudopneumoniae </it>as <it>S. pneumoniae</it>. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.</p

Optimal Control Based Eco-Driving: Theoretical Approach and Practical Applications (Eco-rijden gebaseerd op optimale controle: theoretische aanpak en praktische toepassingen)

Eco-driving is adopting an eco-conscious driving style with the potential to reduce fuel consumption in an easy and cost-effective way. This dissertation develops a methodology to calculate the most fuel-efficient driving behavior. Eco-driving is treated as an optimal control problem. Different low-degree quasistatic polynomial fuel consumption models and optimal control methods are evaluated. The proposed methodology uses quadratic fuel consumption models and Pontryagin s maximum principle. An explicit and comprehensive expression for the optimal engine control is derived. Gear shifting is introduced by maximizing or minimizing an additional variable. Simulations reveal some interesting properties of optimal driving behavior. These results are used to propose refined eco-drive guidelines.status: publishe

Calculation of the minimum-fuel driving control based on Pontryagin’s maximum principle

This paper presents a methodology to calculate the minimum-fuel driving control for a point-mass vehicle on a road with constant slope. The main control is the engine torque. Gear shifting, clutch disengagement, and braking are taken into account with a switching condition. The methodology can take into account distance and time constraints. Only longitudinal vehicle dynamics are used in combination with a quasistatic polynomial fuel consumption model. Simulations are given to analyze the optimal driving control and assess the fuel savings for acceleration and deceleration.publisher: Elsevier articletitle: Calculation of the minimum-fuel driving control based on Pontryagin’s maximum principle journaltitle: Transportation Research Part D: Transport and Environment articlelink: http://dx.doi.org/10.1016/j.trd.2013.05.004 content_type: article copyright: Copyright © 2013 Elsevier Ltd. All rights reserved.status: publishe

Analyis of Eco-Driving Using Polynomial Fuel Consumption Models

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Minimum-fuel control of combustion engine powertrains

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Minimum-fuel Vehicle Driving with Optimal Control

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