粪肠球菌
16S核糖体RNA
微生物学
细菌
实时聚合酶链反应
核糖体RNA
生物
根管
化学
分子生物学
医学
基因
牙科
生物化学
金黄色葡萄球菌
遗传学
作者
Christine Sedgley,Aaron C. Nagel,Charles E. Shelburne,Don B. Clewell,Oliver K. Appelbe,Anders Molander
标识
DOI:10.1016/j.archoralbio.2004.10.017
摘要
Enterococcus faecalis is consistently associated with recurrent root canal infections. Only low concentrations of E. faecalis in the human mouth have been demonstrated by culture techniques. Quantitative detection strategies more sensitive than culturing, such as quantitative PCR (qPCR), could provide more illuminating data. Thirty outpatients attending the University of Michigan Graduate Endodontic Clinic for endodontic treatment provided oral rinse samples that were analysed for E. faecalis using qPCR and microbiological culturing. A SYBR Green I qPCR protocol was developed for the quantifiable detection of E. faecalis and total bacteria in oral rinse samples using primers designed to target the 16S rRNA gene. Annealing temperature and primer, magnesium ion, and dimethyl sulfoxide concentrations were investigated for optimisation of the protocol; a minimum sensitivity limit of 23 rRNA copies (an estimated six E. faecalis cells) was established for E. faecalis in pure culture, and 104 rRNA copies (an estimated 26 E. faecalis cells) in mixed culture. In qPCR assays, based on extrapolation from estimated rRNA gene copy numbers, E. faecalis comprised 0.0006–0.0047% of a total bacteria load that ranged from 5.92 × 105 to 5.69 × 107 cells/ml of oral rinse. E. faecalis was detected in five (17%) samples in concentrations from 114 to 490 cells/ml. In parallel culture assays E. faecalis were detected in only two samples (7%) of the five identified by qPCR and in concentrations 30 and 240 CFU/ml. qPCR reported a higher incidence of E. faecalis in oral rinse samples than culture techniques and afforded greater sensitivity.
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