Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes

TLDR

We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. The method uses a domain‑specific PCR primer set and fluorogenic probe with strong and weak selectivity for archaeal rRNA genes, plus a universal primer set and probe for both bacterial and archaeal rDNAs, enabling rapid, reproducible, and sensitive detection. The system successfully detected and quantified archaeal rDNAs in controlled assemblages and in environmental DNA from both thermophilic and methanogenic habitats, demonstrating its applicability to culture‑independent microbial community analysis.

Abstract

ABSTRACT We describe a rapid, reproducible, and sensitive method for detection and quantification of archaea in naturally occurring microbial communities. A domain-specific PCR primer set and a domain-specific fluorogenic probe having strong and weak selectivity, respectively, for archaeal rRNA genes (rDNAs) were designed. A universal PCR primer set and a universal fluorogenic probe for both bacterial and archaeal rDNAs were also designed. Using these primers and probes, we demonstrated that detection and quantification of archaeal rDNAs in controlled microbial rDNA assemblages can be successfully achieved. The system which we designed was also able to detect and quantify archaeal rDNAs in DNA samples obtained not only from environments in which thermophilic archaea are abundant but also from environments in which methanogenic archaea are abundant. Our findings indicate that this method is applicable to culture-independent molecular analysis of microbial communities in various environments.