Quantitative real-time PCR detection of oral Enterococcus faecalis in humans
Quantitative real-time PCR detection of oral Enterococcus faecalis in humans
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DOI：
10.1016/j.archoralbio.2004.10.017
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发表时间：
2005-06-01
影响因子：
3
通讯作者：
Molander, A
中科院分区：
文献类型：
--
作者：
Sedgley, CM;Nagel, AC;Molander, A
目的：粪肠球菌与根管感染的复发有关。只有两个浓度的E.人类口腔中的粪便已经通过培养技术得到证实。定量检测策略比培养更敏感，如定量PCR（qPCR），可以提供更多的启发data.Design：30名参加密歇根大学研究生根管诊所的根管治疗门诊患者提供的口腔冲洗液样本进行了分析，为E。粪球菌的培养。建立了SYBR绿色I qPCR检测大肠杆菌的方法。粪便和口腔冲洗样品中的总细菌，使用设计用于靶向16 S rRNA基因的引物。退火温度和引物，镁离子和二甲亚砜浓度进行了研究，以优化该协议;最低灵敏度限制为23 rRNA拷贝（估计6 E. faecalis细胞）。faecalis，104个rRNA拷贝（估计26个E.结果：在qPCR检测中，基于从估计的rRNA基因拷贝数外推，E.粪便占总细菌的0.0006-0.0047%，其范围为5.92 × 10（5）至5.69 × 10（7）个细胞/ml口腔冲洗液。E.在5个（17%）样品中检测到粪肠球菌，浓度为114 - 490个细胞/ml。在平行培养试验中，E.在通过qPCR鉴定的5个样品中，仅在浓度为30和240 CFU/ml的2个样品（7%）中检测到粪肠球菌。粪便中的口腔冲洗液样品比培养技术，并提供了更大的灵敏度。© 2004爱思唯尔有限公司版权所有。
Objective: Enterococcus faecalis is consistently associated with recurrent root canal infections. Only tow 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.Design: 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.Results: In qPCR assays, based on extrapolation from estimated rRNA gene copy numbers, E. faecalis comprised 0.0006-0.0047% of a total bacteria toad that ranged from 5.92 x 10(5) to 5.69 x 10(7) 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.Conclusions: qPCR reported a higher incidence of E. faecalis in oral rinse samples than culture techniques and afforded greater sensitivity. © 2004 Elsevier Ltd. All rights reserved.