Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays
Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays
复制标题
DOI：
10.1128/aem.69.5.2423-2429.2003
复制
发表时间：
2003-05-01
影响因子：
4.4
通讯作者：
Conrad, R
中科院分区：
文献类型：
--
作者：
Kolb, S;Knief, C;Conrad, R
土壤中的甲烷氧化主要是由甲烷氧化菌完成的。人们对土壤中甲烷氧化菌的丰度知之甚少，因为通过培养和显微镜技术进行定量非常麻烦。 16S 核糖体 DNA 和 pmoA（（颗粒甲烷单加氧酶的 x 亚基）系统发育树的比较显示出良好的相关性，并揭示了变形菌门的 α 和 γ 亚类内五个不同的甲烷氧化菌组：甲基球菌组、甲基细菌/甲基八叠球菌组、甲基窦菌组、甲基CAPSA 组、 和森林克隆组（从森林土壤中检索的 pmoA 序列簇）。我们通过 SybrGreen 针对这五组以及所有甲烷氧化细菌针对 pmoA 基因开发了定量实时 PCR 检测。所有检测的每个反应的检测限在 10(1) 和 10(2) 个目标分子之间。对添加甲基球菌细胞的土壤样品进行实时 PCR 分析 capsulatus、Mmethylomicrobium album 和 Mmethylosinus trichosporium 回收了几乎所有添加的细菌。只有甲基窦特异性检测仅回收了 20% 的添加细胞，这可能是由于 II 型甲烷氧化菌的裂解效率较低。对淹水稻田土壤中甲烷氧化菌群落结构的分析显示 (5.0 +/- 1.4) X 10(6) pmoA 分子 g(-1) 对于所有甲烷氧化菌。甲基窦组占主导地位（2.7 x 10(6) +/- 1.1 X 10(6) 目标分子 g(-1)）。此外，甲基杆菌/甲基八叠球菌组的细菌丰富（2.0 X 10(6) +/- 0.9 X 10(6) 目标分子 g 土壤(-1)）。另一方面， pmoA 属于森林无性系和甲基壳组，低于 1.9 X 10(4) 目标分子 g 土壤(-1) 的检测限。我们的结果表明，以 pmoA 为目标的实时 PCR 可以对土壤中五种主要的甲烷氧化菌进行快速、灵敏的定量。因此，这种方法将有助于定量分析自然界甲烷氧化菌的群落结构。
Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA ((x subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) X 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 X 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 X 10(6) +/- 0.9 X 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 X 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.