Optimization and application of bacterial environmental DNA and RNA isolation for qualitative and quantitative studies

Abstract

Molecular detection of environmental DNA (eDNA) and RNA (eRNA) allows highly sensitive qualitative (i.e., presence or absence) and quantitative (i.e., abundance) monitoring of aquatic bacteria. However, bacterial molecular diagnostics are limited by low positive predictive values. Protocols for bacterial eDNA and eRNA molecular monitoring have primarily focused on optimizing specimen collection, and the optimal method to purify bacterial nucleic material from postcollection aquatic specimens to maximize the analytical sensitivity of molecular diagnostics remains poorly defined. Accordingly, strategies to isolate bacterial eDNA and eRNA from fresh and saltwater were investigated. We evaluated two filtration and four nucleic acid purification systems as representative of current generation bacterial eDNA and eRNA isolation strategies for capacity to isolate bacterial eDNA and eRNA from prelysed (i.e., free-nucleic acids) and viable (i.e., colony forming units, CFU) bacterial cells. We also compared the sensitivities of reverse transcription quantitative PCR (RT-qPCR) and metagenomic shotgun microbiome sequencing. The optimal protocol used 0.7 μm borosilicate glass filters (Whatman plc) followed by extraction with the RNeasy PowerWater kit (Qiagen). The protocol had a very high analytical sensitivity (10⁻³–10⁰ ng and 10²–10¹ CFU detected in 500 mL) across multiple species of bacteria, when tested with either RTqPCR or metagenomic sequencing. Importantly, this study highlighted several limitations which are restrictive to both qualitative and quantitative bacterial eDNA and eRNA studies. First, a 12-h time course between sampling and extraction revealed significant species-specific changes in cell number and free-nucleic acid concentrations can occur postspecimen collection. Second, we found Gram-positive bacteria yielded less nucleic material compared to Gram-negative bacteria suggesting bacterial eDNA and eRNA studies could be biased by microorganism genome stability and extraction efficiency. This study highlights the need to define the species-specific diagnostic sensitivity of a protocol when monitoring aquatic bacterial eDNA and eRNA with molecular diagnostics.