Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene.
We sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae.
Centre de recherche en infectiologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, Département de microbiologie-infectiologie et d'immunologie, Faculté de médecine, Université Laval, Québec, QC, Canada.
V
V
Sistek
M
M
Boissinot
D K
DK
Boudreau
A
A
Huletsky
F J
FJ
Picard
M G
MG
Bergeron
D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013290:Streptococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013291:Streptococcus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D011938:Rec A Recombinases