A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.
; ; et al
in Applied Microbiology and Biotechnology (2016), 100(3), 1377-1392
Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into ... [more ▼]
Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR(R)green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR(R)green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days. [less ▲]
Development and performance assessment of a qualitative SYBR(R) green real-time PCR assay for the detection of Aspergillus versicolor in indoor air.
; ; et al
in Applied Microbiology and Biotechnology (2015), 99(17), 7267-7282
Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding ... [more ▼]
Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR(R) green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR(R) green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods. [less ▲]
Microbial Diversity and Processes in Lake Kivu (East Africa)
; Darchambeau, François ; et al
Lake Kivu is a deep meromictic and oligotrophic tropical African lake with a permanent thermal- and haline stratification with huge accumulations of dissolved CO2 and CH4 (ca. 300 km3 and 60 km3 ... [more ▼]
Lake Kivu is a deep meromictic and oligotrophic tropical African lake with a permanent thermal- and haline stratification with huge accumulations of dissolved CO2 and CH4 (ca. 300 km3 and 60 km3, respectively) in the deep anoxic monimolimnion (from 60 o 480 m depth). Although there are a wealth of information on the ecology of small eukaryotes and their trophic role on Kivu, available information on prokaryotic planktonic assemblages is scarce. Molecular analysis of archaeal and bacterial communities showed a vertical segregation imposed by the permanent redoxcline. In relation to Bacteria, Actinobacteria, Betaproteobacteria, Green Sulfur Bacteria and Bacteroidetes were the most commonly retrieved groups. For Archaea, a marked dominance of Thaumarchaeota and Crenarchaeota (75% of all archaeal OTUs) over Euryarchaeota was observed. In the anoxic hypolimnion, Euryarchaoeta (Methanosarcinales and Methanocellales) lineages together with Miscellaneous Crenarchaeotic Group phylotypes were mainly recovered. In turn, Thaumarchaeota phylotypes were recovered in oxic and suboxic waters. CARDFISH analyses over the first 100 m revealed the dominance of Bacteria (51.4% – 95.7% of DAPI-stained cells), especially Actinobacteria (epilimnion), Betaproteobacteria (oxic-anoxic interface) and Bacteroidetes (upper hypolimnion), over Archaea (1.0% – 4.5%; maximum abundances at the oxic-anoxic interface). In turn, flow cytometry evidenced the dominance of HNA cells in the euphotic layer, whereas the proportion of LNA cells increased with depth. HNA and LNA populations were still observed in the anoxic hypolimnion suggesting facultative or strict anaerobic metabolisms. The detection of distinct depth maxima of nitrate, nitrite, archaeal amoA and Marine Thaumarchaeota 16S gene copy numbers together with regularly detection of deep maxima of 3H-Thymidine uptake, and the presence of low-light adapted GSB species point towards a strong link of N, C, and S cycles in the redoxcline of Lake Kivu. [less ▲]Detailed reference viewed: 67 (2 ULg)