Open Access Highly Accessed Research article

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.

António Machado1, Carina Almeida12, Débora Salgueiro1, Ana Henriques1, Mario Vaneechoutte3, Freddy Haesebrouck4, Maria João Vieira1, Ligia Rodrigues1, Nuno Filipe Azevedo2 and Nuno Cerca1*

Author Affiliations

1 IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga, 4710-057, Portugal

2 LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, rua Dr. Roberto Frias, Porto, 4200-465, Portugal

3 Laboratory of Bacteriology Research, Faculty Medicine & Health Sciences, University of Ghent, Ghent, B-9000, Belgium

4 Laboratory of Veterinary Bacteriology and Mycology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, B9820, Belgium

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BMC Microbiology 2013, 13:82  doi:10.1186/1471-2180-13-82

Published: 12 April 2013

Abstract

Background

Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples.

Results

Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.

Conclusions

This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

Keywords:
Fluorescence in situ Hybridization (FISH); Peptide Nucleic Acid Probe (PNA probe); Lactobacillus spp.; Gardnerella vaginalis; Bacterial vaginosis