Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.
In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.
LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal; Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark; ICBAS, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal.
Sílvia
S
Fontenete
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Marina
M
Leite
LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal; Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.
Nuno
N
Guimarães
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; ICBAS, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal; IBMC, Institute for Molecular Biology and Cell Biology, Porto, Portugal.
Pedro
P
Madureira
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Rui Manuel
RM
Ferreira
Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal; FMUP, Faculty of Medicine of Porto University, Porto, Portugal.
Céu
C
Figueiredo
IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Jesper
J
Wengel
LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
Nuno Filipe
NF
Azevedo
D005434:Flow Cytometry; D005753:Gastric Mucosa / Q000382:microbiology*; D016481:Helicobacter Infections / Q000175:diagnosis*; D016480:Helicobacter pylori; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000378:metabolism*
D009841:Oligonucleotides; C477371:locked nucleic acid