Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method
Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method
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DOI：
10.1016/j.femsim.2004.08.005
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发表时间：
2005-02-01
影响因子：
--
通讯作者：
Takashiba, S
中科院分区：
文献类型：
--
作者：
Maeda, H;Kokeguchi, S;Takashiba, S
采用一种核酸扩增技术--环介导恒温扩增(LAMP)技术，建立了一种快速、简便的牙周病原菌-牙龈卟啉单胞菌检测系统。设计了一套针对16S核糖体RNA基因的6条引物。利用该检测系统，在64℃恒温条件下，30分钟内扩增出目的DNA并显示在琼脂糖凝胶上，检出限为20个牙龈假单胞菌。在没有凝胶电泳的情况下，通过添加SYBR Green I直接在反应管中显示灯扩增子以进行肉眼检查。LAMP反应还通过试管中焦磷酸镁(LAMP的副产物)的白色浑浊度来评估。这些肉眼检查的检测下限分别为20个细胞和200个细胞。虽然从107多个牙髓卟啉单胞菌细胞中观察到假阳性DNA扩增，但在其他5种相关口腔病原体中未观察到扩增。此外，通过使用SYBR Green I在10(2)-10(6)个细胞范围内线性地实时监测LAMP反应来完成对牙龈假单胞菌的定量检测。然后将实时LAMP应用于牙菌斑的临床样本，结果显示与传统的实时荧光PCR结果几乎相同，且具有快速的优势。这些发现表明LAMP在检测和定量牙龈假单胞菌方面具有潜在的实用价值，特别是在其快速和简单方面。(C)2004年欧洲微生物学会联合会。爱思唯尔出版，版权所有。
A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64degreesC with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 107 cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.