Specific magnetic bead based capture of genomic DNA from clinical samples: application to the detection of group B streptococci in vaginal/anal swabs.
Clin Chem
; 53(9): 1570-6, 2007 Sep.
Article
em En
| MEDLINE
| ID: mdl-17660271
ABSTRACT
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.CONCLUSION:
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.
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Base de dados:
MEDLINE
Assunto principal:
Canal Anal
/
Streptococcus agalactiae
/
Vagina
/
DNA Bacteriano
/
Genoma Bacteriano
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article