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Quantification of endospore-forming Firmicutes by qPCR with the functional gene spo0A

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Bueche Matthieu, Wunderlin Tina, Roussel-Delif Ludovic, Junier Thomas, Sauvain Loic, Jeanneret Nicole, Junier Pilar,
Project Metabolic capabilities of spore-forming microorganisms
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Original article (peer-reviewed)

Journal Applied and Environmental Microbiology
Volume (Issue) 79(17)
Page(s) 5302 - 5312
Title of proceedings Applied and Environmental Microbiology
DOI 10.1128/aem.01376-13

Abstract

Bacterial endospores are highly specialized cellular forms that allow Endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR. For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated, given this method a promising potential for the detection of EFF in a wide range of applications.
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