In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp.

Ana Henriques,Tatiana Cereija,Nuno Cerca,António Machado

doi:10.1186/1756-0500-5-637

Ana Henriques, Tatiana Cereija + Show 2 more

Open Access

https://doi.org/10.1186/1756-0500-5-637

Copy DOI

Abstract

BackgroundBacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used.FindingsHere we demonstrate that in silico analytical testing of primer specificity is not a synonym to in vitro analytical specificity by testing a range of published and newly designed primers with both techniques for the detection of BV-associated microorganisms.ConclusionsBy testing primer in vitro specificity with a sufficient range of bacterial strains, we were able to design primers with higher specificity and sensitivity. Also by comparing the results obtained for the newly designed primers with other previously published primers, we confirmed that in silico analysis is not sufficient to predict in vitro specificity. As such care must be taken when choosing the primers for a detection assay.

Highlights

Bacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR
It has been recognized that this pathology is caused by a shift in the microbial ecosystem colonizing the vagina of healthy women; from a Lactobacillus spp. dominated microbial population to the proliferation of other anaerobic microorganisms such as Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp. among others [2,3,4]
It has been described that G. vaginalis may account for 60 to 90% of the BV biofilm mass, while A. vaginae may

Summary

Introduction

Bacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. This technique poses some limitations, already described for other applications, namelly significant differences between the in silico prediction for primer specificity/sensitivity and the actual in vitro results [8,9].

Results

Conclusion