Date Published: May 31, 2019
Publisher: Public Library of Science
Author(s): Andreia S. Azevedo, Inês M. Sousa, Ricardo M. Fernandes, Nuno F. Azevedo, Carina Almeida, Irina V. Lebedeva.
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.
Fluorescence in situ hybridization (FISH) is one of the most well-established molecular biology techniques used for the rapid and direct detection, localization and quantification of microorganisms in many fields of microbiology (e.g. [1–8]). This technique is based on the hybridization of fluorescently-labeled molecules (also called probes) with the conserved 16S or 23S rRNA sequences, particularly abundant and relatively stable in viable cells .
Despite the successful applications of LNA/2′OMe-FISH in bacterial identification (e.g. [4, 44, 45]), there is an absence of studies that assess the impact of denaturant and salt concentration on its efficiency. Hybridization temperature was also modeled in order to assess an optimal range for a standard LNA/2′OMe-probe design (probes with ~14 nucleotides, 64% of GC content, one LNA at very third position of the 2′OMe and phosphorothioate backbones). However, the size and number of residues are aspects that will have great impact on the probe temperature range. This information is very important to find the more suitable hybridization conditions for bacteria detection and to move towards a tailored design of hybridization experiments.
This work follows a set of optimization works that our research group has developed (e.g. [37, 38, 42]) to try to understand the parameters that are essential for the hybridization kinetics of nucleic acid mimics; so that we can move from a trial and error approach to a systematic optimization. While this study can provide a guide for further optimizations and help on establishing new LNA/2’OMe-FISH protocols, the optimal hybridization conditions will depend on the researchers’ goal. For instance, if a multiplex is expected, the conditions should be moved toward the species with weak signal to balance the overall outcome.