Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reactions and a novel bioluminescent pyrophosphate assay
Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reactions and a novel bioluminescent pyrophosphate assay
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DOI：
10.1016/j.ab.2004.06.026
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发表时间：
2004-10-15
影响因子：
2.9
通讯作者：
Maeda, M
中科院分区：
文献类型：
--
作者：
Arakawa, H;Karasawa, K;Maeda, M
我们开发了一种新型生物发光测定法，用于检测聚合酶链式反应 (PCR) 产品中的焦磷酸盐。该方法的原理是：在底物丙酮酸磷酸和辅酶5'-单磷酸腺苷存在下，通过丙酮酸磷酸二激酶将PCR释放的焦磷酸转化为5'-三磷酸腺苷(ATP)；随后，通过萤火虫荧光素酶反应测定 ATP 浓度。焦磷酸盐的检测限为 1.56 x 10(-15) mol/测定。此外，发光强度在大约100秒时达到最大值，并在10分钟后保持较高水平。该方法适用于牙菌斑中致龋菌的检测。因此，通过所提出的生物发光测定法测量了本研究中开发的变形链球菌和远缘链球菌的等位基因特异性 PCR 产物。该方案不需要昂贵的设备，可用于快速监测牙菌斑中的致龋细菌。 (C) 2004 Elsevier Inc. 保留所有权利。
We developed a novel bioluminescent assay for detection of pyrophosphate in polymerase chain reaction (PCR) product. The principle of this method is as follows: pyrophosphate released by PCR is converted to adenosine 5'-triphosphate (ATP) by pyruvate phosphate dikinase in the presence of the substrate pyruvate phosphate and the coenzyme adenosine 5'-monophosphate; subsequently, ATP concentration is determined by firefly luciferase reaction. The detection limit of pyrophosphate is 1.56 x 10(-15) mol/assay. Additionally, luminescent intensity reached a maximum at similar to100s and remained elevated beyond 10min. This approach is applicable to the detection of cariogenic bacteria in dental plaque. Thus, the allele-specific PCR products of Streptococcus mutans and Streptococcus sobrinus developed in this study were measured via the proposed bioluminescent assay. This protocol, which does not require expensive equipment, can be utilized to rapidly monitor cariogenic bacteria in dental plaque. (C) 2004 Elsevier Inc. All rights reserved.