Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy
Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy
复制标题
DOI：
10.1111/j.1574-6968.2008.01119.x
复制
发表时间：
2008-05-01
影响因子：
2.1
通讯作者：
Ovreas, Lise
中科院分区：
文献类型：
--
作者：
Einen, Jorn;Thorseth, Ingunn H.;Ovreas, Lise
本文介绍了一种SYBR Green实时定量PCR（Q - PCR）检测方法，用于检测和定量不同年龄和水深的海底玄武岩玻璃质外壳中存在的细菌和古菌。设计了两组特定域的引物，并对从玄武岩玻璃中提取的DNA中的细菌和古菌16S rRNA基因进行特异性检测和定量验证。还通过对SYBR Gold染色样本的荧光显微镜分析估计了总细胞数。两种不同方法的结果是一致的，Q - PCR结果显示玄武岩中存在的细胞总数在每克玄武岩玻璃6×10⁵到4×10⁶个细胞的范围内。此外，还证明了这些细胞几乎完全来自细菌域。当对不同年龄（22年 - 0.1百万年）和水深（海平面以下139 - 3390米）的样本应用相同方法时，未检测到细胞浓度或古菌和细菌相对丰度的显著差异。
A SYBR Green real-time quantitative PCR (Q-PCR) assay for the detection and quantification of Bacteria and Archaea present in the glassy rind of seafloor basalts of different ages and water depths is presented. Two sets of domain-specific primers were designed and validated for specific detection and quantification of bacterial and archaeal 16S rRNA genes in DNA extracted from basaltic glass. Total cell numbers were also estimated by fluorescence microscopy analysis of SYBR Gold-stained samples. The results from the two different approaches were concurrent, and Q-PCR results showed that the total number of cells present in basalts was in the range from 6 x 10(5) to 4 x 10(6) cells g(-1) basaltic glass. Further, it was demonstrated that these cells were almost exclusively from the domain Bacteria. When applying the same methods on samples of different ages (22 years-0.1 Ma) and water depths (139-3390 mbsl), no significant differences in cell concentrations or in the relative abundance of Archaea and Bacteria were detected.