Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

Sílvia Fontenete,Nuno Guimarães,Jesper Wengel,Nuno Filipe Azevedo,Céu Figueiredo,Marina Leite,Rakesh N Veedu

doi:10.1371/journal.pone.0081230

Abstract

The understanding of the human microbiome and its influence upon human life has long been a subject of study. Hence, methods that allow the direct detection and visualization of microorganisms and microbial consortia (e.g. biofilms) within the human body would be invaluable. In here, we assessed the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2’-O-methyl RNAs (2’OMe) with two types of backbone linkages (phosphate or phosphorothioate), we were able to successfully identify two probes that hybridize at 37 °C with high specificity and sensitivity for H. pylori, both in pure cultures and in gastric biopsies. Furthermore, the use of this type of probes implied that toxic compounds typically used in FISH were either found to be unnecessary or could be replaced by a non-toxic substitute. We show here for the first time that the use of advanced LNA probes in FIVH conditions provides an accurate, simple and fast method for H. pylori detection and location, which could be used in the future for potential in vivo applications either for this microorganism or for others.

Highlights

The human microbiome has long been studied for a better understanding of its influence upon human development, physiology, immunity, and nutrition [1]
The initial purpose of this work was to find a type of synthetic oligonucleotide that would be capable of efficiently hybridizing in a bacterium at human body temperature (37 °C)
After a biophysics analysis comparing melting and hybridization temperatures of the locked nucleic acids (LNA) probes, we concluded that the hybridization temperature should be between 15-30 °C lower than the melting temperature measured under similar conditions

Summary

Introduction

The human microbiome has long been studied for a better understanding of its influence upon human development, physiology, immunity, and nutrition [1] In most of these studies, microbial identification methods rely on sample collection followed by DNA isolation and sequencing [2,3]. Fluorescent in situ hybridization (FISH) using DNA probes has long been used to rapidly detect and localize microbial cells in human clinical samples [4,5]. This method was never employed to detect microorganisms within the human body (or other higher-order animals).

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