Parham Nicholas J, Picard François J, Peytavi Régis, Gagnon Martin, Seyrig Grégoire, Gagné Pier-Ann, Boissinot Maurice, Bergeron Michel G
Centre de Recherche en Infectiologie de l'Université Laval, Centre Hospitalier Universitaire de Québec (Pavillon CHUL), Québec, Canada.
Clin Chem. 2007 Sep;53(9):1570-6. doi: 10.1373/clinchem.2007.091389. Epub 2007 Jul 27.
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.
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.
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.
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.
B族链球菌（GBS）是新生儿败血症和脑膜炎的主要病因。我们之前开发了一种用于从分娩期间孕妇的阴道/肛门样本中检测GBS的快速诊断系统。为便于将该方法应用于即时检测，我们开发了一种特异性且高效的GBS DNA捕获方法，该方法与PCR和非扩增检测技术均兼容。
用不同长度的寡核苷酸捕获探针将超顺磁性微珠功能化，用于捕获GBS基因组DNA（gDNA）。采用快速提取程序从GBS培养物或添加了GBS的阴道/肛门样本中提供DNA。在30 μL杂交缓冲液中，将功能化微珠与靶DNA进行杂交反应，在室温下进行1小时，随后洗涤并重悬于水中。然后使用定量PCR检测捕获的DNA。
一个25聚体捕获探针能够检测到1000个纯化GBS DNA基因组拷贝。使用50聚体（100个拷贝）和70聚体捕获探针（10个拷贝）提高了检测GBS的能力。对于稀释的GBS肉汤培养物以及添加了GBS的阴道/肛门拭子样本，实现了约1250 CFU/mL的检测。
寡核苷酸功能化的超顺磁性微珠能有效地从细菌培养物以及添加了GBS的阴道/肛门样本中捕获GBS gDNA。gDNA捕获效率随寡核苷酸长度增加而提高。该技术可与微流控系统中的样品制备和检测技术相结合，实现GBS的即时检测。
