title: In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp.
abstract: BACKGROUND
  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. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  By 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.
authors.affiliation: CEB-IBB, Centro de Engenharia Biológica - Instituto de Biotecnologia e Bioengenharia, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Henriques
authors.forename: Tatiana
authors.initials: T
authors.lastname: Cereija
authors.forename: António
authors.initials: A
authors.lastname: Machado
authors.forename: Nuno
authors.initials: N
authors.lastname: Cerca
mesh_terms: D039903:Actinobacteria / Q000235:genetics*; D000328:Adult; D017931:DNA Primers / Q000235:genetics*; D030561:Databases, Nucleic Acid; D005260:Female; D006194:Gardnerella vaginalis / Q000235:genetics*; D006801:Humans; D007778:Lactobacillus / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013045:Species Specificity; D016585:Vaginosis, Bacterial / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D017931:DNA Primers