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 procedures for RNA-extraction from peripheral blood mononuclear cells

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit–blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs

Development of a qPCR platform for quantification of the five bacteriophages within bacteriophage cocktail 2 (BFC2)

To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO

Evaluation of the Stability of Bacteriophages in Different Solutions Suitable for the Production of Magistral Preparations in Belgium

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6-9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days

Kruisreactiviteit van monoclonale antilichamen met lever-metallothioneines van zoogdieren

KULeuven Campusbibliotheek Exacte Wetenschappen / UCL - Université Catholique de LouvainSIGLEBEBelgiu

Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA)

Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification tool for distinguishing between 16 Candida spp., i.e. Candida albicans, Candida bracarensis, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lipolytica, Candida lusitaniae, Candida nivariensis, Candida norvegensis, Candida parapsilosis, Candida tropicalis and Candida sojae, and Saccharomyces cerevisiae and one species pair, i.e. Candida metapsilosis/Candida orthopsilosis. Starting from a cultured isolate, ITS2-MCA led to differentiation of these species within 6 h. According to our findings, ITS2-MCA offers a simple, rapid and cost-effective method for identification of cultured isolates of the clinically most relevant and prevalent Candida species. Further studies will be necessary to evaluate how it performs on mixed samples and clinical samples

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

Abstract 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 μl sample in a final volume of 10 μl 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.</p