Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids

Roland Martzy,Katharina Bica-Schröder,Claudia Kolm,Robert L Mach,Georg H Reischer,Ádám Márk Pálvölgyi,Regina Sommer,Rudolf Krska,Andreas H Farnleitner,Stefan Jakwerth,Alexander K T Kirschner

doi:10.1038/s41598-019-50246-5

Abstract

The extraction of nucleic acids from microorganisms for subsequent molecular diagnostic applications is still a tedious and time-consuming procedure. We developed a method for the rapid preparation of genomic DNA from bacteria based on hydrophilic ionic liquids (ILs). First, we tested eight ILs in different buffer systems for their inhibitory effects on quantitative PCR. The cell lysis potential of different IL/buffer combinations was assessed by application on Enterococcus faecalis as a model organism for Gram-positive bacteria. The two best ILs, choline hexanoate and 1-ethyl-3-methylimidazolium acetate, were compared with the reference enzymatic method and two commercial DNA extraction kits. All methods were evaluated on four Gram-positive and four Gram-negative bacterial species that are highly relevant for environmental, food, or clinical diagnostics. In comparison to the reference method, extraction yields of the IL-based procedure were within one order of magnitude for most of the strains. The final protocol for DNA extraction using the two ILs is very low-cost, avoids the use of hazardous chemicals and can be performed in five minutes on a simple heating block. This makes the method ideal for high sample throughput and offers the opportunity for DNA extraction from bacteria in resource-limited settings or even in the field.

Highlights

The field of microbial molecular diagnostics comprises various methods for the specific detection of nucleic acids (NAs) from different microorganisms1,2 – e.g., human pathogens in clinical and environmental samples[3,4,5], faecal indicator bacteria in water[6,7], or harmful microbial agents in food and feed[8,9]
A more efficient and user-friendly DNA extraction method could promote the progress of molecular point-of-care detection methods, which are still dependent on sophisticated laboratory infrastructure
The cell wall of Gram-positive bacteria is protected by a hardy peptidoglycan layer[14], which remained unaffected by the treatment with hydrophobic ionic liquids (ILs) and high temperatures[14]

Summary

Introduction

The field of microbial molecular diagnostics comprises various methods for the specific detection of nucleic acids (NAs) from different microorganisms1,2 – e.g., human pathogens in clinical and environmental samples[3,4,5], faecal indicator bacteria in water[6,7], or harmful microbial agents in food and feed[8,9]. State-of-the-art extraction procedures for microorganisms traditionally use incubation steps with enzymes such as lysozyme and proteinase K to digest cell wall components and interfering proteins, respectively Hazardous chemicals such as phenol and chloroform[10], or commercial kits are used for NA purification, depending on the area of application and the matrix in which the cells are investigated. The aim of this study was the development of a novel IL-based method for the rapid lysis of Gram-positive bacteria that can be carried out with minimal laboratory equipment for subsequent DNA-based diagnostics To this end, we selected Enterococcus faecalis as a model organism and tested a selection of hydrophilic ionic liquids on their cell lysis potential. After optimizing the reaction conditions regarding buffer system, IL concentration, temperature and incubation time, the novel DNA preparation method was compared to state-of-the-art protocols and commercial DNA extraction kits with different bacterial targets

Objectives

Methods

Results