A rapid method for extraction and purification of DNA from dental plaque.
A rapid method for extraction and purification of DNA from dental plaque.
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一种从牙菌斑中提取和纯化 DNA 的快速方法。
DOI：
10.1128/aem.61.11.4120-4123.1995
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发表时间：
1995
影响因子：
4.4
通讯作者：
Greenberg,EP
中科院分区：
文献类型：
--
作者：
Parrish,KD;Greenberg,EP
基于先前描述的DNA提取程序的快速方法被开发用于从牙菌斑样品中分离DNA。分离的DNA适用于PCR。使用冻融、细胞壁降解酶和异硫氰酸胍裂解细胞并释放DNA。将释放的DNA吸附到硅藻土上，并通过用异硫氰酸胍、乙醇和丙酮洗涤来纯化。将纯化的DNA从硅藻土释放到水性缓冲液中，并通过PCR用16S rDNA引物（rDNA是编码rRNA的DNA）进行分析。根据对许多细菌菌种的纯培养物的研究判断，革兰氏阴性和革兰氏阳性微生物通过该程序同样良好地裂解。PCR产物的量与在500至50，000个细胞的测试范围内分析的细胞数量成比例。根据对加标已知量口腔细菌齿垢密螺旋体的菌斑样本的研究，从菌斑制备的DNA不含抑制PCR的物质。这种方法不仅在未培养的菌斑样品中，而且在其他复杂的细菌组合中的细菌种群的分子遗传学研究中应具有实用性。
A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.