Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air

Abstract

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® 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® 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.

Discrimination of three genetically close Aspergillus species by using high resolution melting analysis applied to indoor air as case study

“…For example, we previously proposed a qualitative qPCR SYBR®Green method targeting the ITS2 region ( Aversi_ITS assay) for the detection of Aspergillus versicolor [13] , an important indoor fungal contaminant [2, 14, 15]. Among the 10 species from the indoor air background that had been included in the specificity test, this tool developed for the specific detection of A. versicolor resulted in false positives only for the DNA of 2 genetically close species, i.e., Aspergillus creber and Aspergillus sydowii , belonging to the same group of Versicolores .…”

“…As previously elaborated [13], these 3 species are difficult to be discriminated, both morphologically as genetically. The available Taqman assays of the United States Environmental Protection Agency (EPA) for the specific detection of A. versicolor and A. sydowii respectively, also amplify both species each time [11, 16].…”

“…In the present study, we developed a molecular method based on our previously proposed SYBR®Green qPCR method for the detection of A. versicolor [13], for the discrimination of A. versicolor , A. sydowii and A. creber . Indeed, the advantage of SYBR®Green includes the possibility to discriminate different amplicons based on their melting temperature (T m ).…”

Development and performance assessment of a luminex xMAP® direct hybridization assay for the detection and identification of indoor air fungal contamination

“…sydowii and A . versicolor ) species was already observed in other studies [10, 15]. To improve the specific identification of these 3 species, an additional marker could be added such as the gene coding for β-tubulin or mycotoxin genes [20, 42].…”

“…The protocols for the sampling, the DNA extraction and microscopic determination were previously described in Libert et al (2015) [10]. All the samples were collected in duplicate.…”