Quantification of anaerobic methanotrophic archaea Candidatus Methanoperedens of the ANME-2d cluster using new PCR primers

Abstract

Archaea related to Candidatus Methanoperedens previously classified as anaerobic methanotrophs (ANME-2d), play critical roles in the anaerobic oxidation of methane in terrestrial environments. Despite their presumed widespread distribution, the ability of primers to detect them in environmental samples is often insufficient because of low sensitivity and specificity. We therefore developed new primers ANME2d664F and ANME2d850R that are specific for 16S rRNA gene of Ca. Methanoperedens. In silico analyses suggest that these primers are likely to preferentially detect Ca. Methanoperedens-related sequences. We validated their performance in environmental samples in which Ca. Methanoperedens had previously been detected, enabling consistent detection across sediment depth profiles. The quantitative PCR results revealed that the range of Ca. Methanoperedens abundance was 3.6 × 10⁶-9.8 × 10⁷ genes/g of sediment, and that the abundance peaked at a depth of 7 cm. Our results showed that our primer set is a useful tool for investigating the distribution and potential ecological functions of archaea related to Ca. Methanoperedens in methane-cycling environments.

Keywords: Anaerobic methane oxidation; Candidatus Methanoperedens; Primer; qPCR.