ArticlePloS one2019
Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.
Article in PloS one, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 23 citations in OpenAlex.
- Targeting intestinal inflammation using locked nucleic acids delivered via lipid nanoparticles.Nature communications · 2025Article
- Research advances of the establishment and characterization ofFrontiers in microbiology · 2025Review
- Assessment of UTI Diagnostic Techniques Using the Fuzzy-PROMETHEE Model.Diagnostics (Basel, Switzerland) · 2023Article
- Recent Advances in High-sensitivityActa histochemica et cytochemica · 2023Review
- Development of a Novel Peptide Nucleic Acid Probe for the Detection ofMicroorganisms · 2022Article
- Spectral imaging and nucleic acid mimics fluorescenceFrontiers in microbiology · 2022Article
- Microfluidics combined with fluorescenceFrontiers in bioengineering and biotechnology · 2022Article
- Friends with Benefits: An Inside Look of Periodontal Microbes' Interactions Using Fluorescence In Situ Hybridization-Scoping Review.Microorganisms · 2021Article
- Lipoplexes to Deliver Oligonucleotides in Gram-Positive and Gram-Negative Bacteria: Towards Treatment of Blood Infections.Pharmaceutics · 2021Article
- Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?Antibiotics (Basel, Switzerland) · 2021Article
- An Introduction to Fluorescence in situ Hybridization in Microorganisms.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Flow-FISH Using Nucleic Acid Mimic Probes for the Detection of Bacteria.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Application of Nucleic Acid Mimics in Fluorescence In Situ Hybridization.Methods in molecular biology (Clifton, N.J.) · 2021Article
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
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Abstract
Despite the successful application of LNA/2'OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2'OMe hybridization step-hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2'OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2'OMe and 64% of GC content, should be use in initial optimization of new LNA/2'OMe-FISH protocols.
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