Specific magnetic bead based capture of genomic DNA from clinical samples: application to the detection of group B streptococci in vaginal/anal swabs.
BACKGROUND
Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns. We previously developed a rapid diagnostic system for GBS detection from vaginal/anal samples obtained from pregnant women during delivery. To facilitate the adaptation of this method for point-of-care testing, we have developed a specific and efficient GBS DNA capture method that is compatible with both PCR and nonamplification detection technologies.


METHODS
Superparamagnetic beads were functionalized with oligonucleotide capture probes of different lengths and used to capture GBS genomic DNA (gDNA). A rapid extraction procedure was used to provide DNA from GBS cultures or vaginal/anal samples with added GBS. Hybridization reactions consisting of functionalized beads and target DNA in 30 muL of hybridization buffer were performed for 1 h at room temperature, followed by washing and resuspension in water. Captured DNA was then detected using quantitative PCR.


RESULTS
A 25-mer capture probe allowed detection of 1000 genome copies of purified GBS DNA. The ability to detect GBS was improved by use of a 50-mer (100 copies) and a 70-mer capture probe (10 copies). Detection of approximately 1250 CFU/mL was achieved for diluted GBS broth culture and for vaginal/anal swab samples with added GBS.


CONCLUSIONS
Oligonucleotide-functionalized superparamagnetic microbeads efficiently capture GBS gDNA from both bacterial cultures and vaginal/anal samples with added GBS. Efficiency of gDNA capture increases with oligonucleotide length. This technology could be combined with sample preparation and detection technologies in a microfluidic system to allow point-of-care testing for GBS.
Parham
Nicholas J
NJ
Centre de Recherche en Infectiologie de l'Université Laval, Centre Hospitalier Universitaire de Québec (Pavillon CHUL), Québec, Canada.
Picard
François J
FJ
Peytavi
Régis
R
Gagnon
Martin
M
Seyrig
Grégoire
G
Gagné
Pier-Ann
PA
Boissinot
Maurice
M
Bergeron
Michel G
MG
D001003:Anal Canal / Q000382:microbiology*; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000032:analysis*; D036861:Delivery, Obstetric; D005260:Female; D016680:Genome, Bacterial; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D019095:Point-of-Care Systems; D011247:Pregnancy; D013292:Streptococcus agalactiae / Q000235:genetics* / Q000302:isolation & purification; D014621:Vagina / Q000382:microbiology*
D004269:DNA, Bacterial