Development of an absolute quantification method for ribosomal RNA gene copy numbers per eukaryotic single cell by digital PCR

Abstract

Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.