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

Abstract

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®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®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.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes

“…Exophiala is a member of the ascomycete order Chaetothyriales (fungi), comprising the black yeasts and allies, which are frequently encountered as causative agents of disorders in humans and animals [1][2][3][4][5][6][7]. Human infections vary from commensalism or moderate cutaneous infection to fatal neurotropism with serious mutilation.…”

Exploiting the Advantages of Molecular Tools for the Monitoring of Fungal Indoor Air Contamination: First Detection of Exophiala jeanselmei in Indoor Air of Air-Conditioned Offices

“…The protocol used for the classical analysis (i.e., culture and fungal determination) of the water and air samples, collected with the Coriolis ® µ air sampler, has been previously described [31][32][33]. According to the protocols, the incubation time ranged between five and 21 days as recommended for the detection of major indoor air species and for the detection of E. jeanselmei.…”

“…The protocol used for the DNA extraction from water [31] and indoor air samples [33] was previously described. This extraction protocols have been optimized in order to avoid inhibition in downstream PCR-based analysis.…”

“…The analyses of DNA extracted from air and water samples were performed in duplicate in two independent runs. The RT-PCR assay used was the SYBR ® Green Ejeanselmei_ITS assay earlier reported for the detection of E. jeanselmei in water [31]. This assay uses the SYBR ® green PCR Mastermix (Diagenode, Liège, Belgium).…”

“…This assay uses the SYBR ® green PCR Mastermix (Diagenode, Liège, Belgium). The sequences of the primers used (targeting the internal transcribed spacer region (ITS)) are provided by Libert and colleagues [31]. The primers were purchased from Eurogentec (Liège, Belgium).…”