Almeida Carina, Constante Diogo, Ferreira André, Cerqueira Laura, Vieira Maria J, Azevedo Nuno F
LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.
Future Microbiol. 2014;9(10):1131-42. doi: 10.2217/fmb.14.68.
Developments on synthetic molecules, such as peptide nucleic acid (PNA), make FISH procedures more robust for microbial identification. Fluorochromes use might hinder a broader implementation of PNA-FISH, but colorimetric applications are inexistent so far.
A biotin-labeled eubacteria probe was used to develop a colorimetric PNA-in situ hybridization (ISH) assay. An enzymatic-conjugate, targeting biotin, was introduced. The procedure was optimized and evaluated regarding sensitivity, specificity and detection limit.
RESULTS have shown strong ISH signals. The method was specific, but permeabilization problems were observed for Gram-positive bacteria. Detection limit was 5 × 10(7) CFU/ml, limiting current applications to pre-enriched samples.
The PNA-ISH procedure described here is a simple alternative to other detection methods, and is also the base for the development of other PNA colorimetric systems.
