Institute of Microbiology, Leopold-Franzens University Innsbruck, Technikerstr. 25, 6020, Innsbruck, Austria,
World J Microbiol Biotechnol. 2014 Jan;30(1):293-304. doi: 10.1007/s11274-013-1450-x. Epub 2013 Aug 6.
In the present study nine promising primer sets, targeting Archaea and methanogenic Archaea in particular, were evaluated in silico, in vitro and in situ concerning specificity, accuracy and applicability in end-point (ep-) and especially quantitative (q-)PCR research. The main goal was to adapt and evaluate already adapted primer sets, which were partially designed in combination with TaqMan probes, in substantially cheaper SYBR Green-based qPCR applications. An initial 16S rRNA gene bank-based in silico evaluation revealed high coverage potentials for all primers within targeted groups, ranging from 71 to 90%, except the Methanosaeta specific set showing a low potential of 37%. Mentionable cross-reacting potentials could be detected for the Methanothermobacter, Methanomicrobiales and Methanoculleus sets. The in vitro evaluation with selected reference organisms revealed a specific behavior for most primer sets, while the Methanosarcina and Methanothermobacter sets showed most problematic cross-reactions in epPCR application. We were able to show that primers for detecting the total archaeal community, methanogenic orders Methanosarcinales, Methanobacteriales, Methanococcales and the genus Methanoculleus performed in a highly specific way and allowed an accurate quantification of targeted organisms without the use of expensive TaqMan probes. However, primer pairs designed for detecting Methanomicrobiales, Methanothermobacter, Methanosarcina and Methanosaeta are not suitable for SYBR Green applications. The reliability of in situ quantifications was assessed for a typical methanogenic community, derived from a thermophilic fermenter, and confirmed via denaturing gradient gel band quantification and sequencing. Thereby, we revealed high abundances of methanogenic Archaea, mainly comprising Methanoculleus and Methanosarcinales, while Methanobacteriales only formed a minor fraction.
在本研究中，针对古菌特别是产甲烷古菌，评估了九种有前景的引物，包括特异性、准确性和适用性，包括终点（ep-）和定量（q-）PCR 研究。主要目标是适应和评估已经适应的引物，这些引物部分与 TaqMan 探针结合设计，应用于成本更低的 SYBR Green 基础 qPCR 应用中。初步的基于 16S rRNA 基因库的计算机评估显示，所有针对目标群体的引物都具有很高的覆盖率潜力，范围从 71%到 90%，除了 Methanosaeta 特异性引物组的低覆盖率潜力 37%。可检测到 Methanothermobacter、Methanomicrobiales 和 Methanoculleus 组的交叉反应潜力。选择参考生物体的体外评估显示，大多数引物组的行为具有特异性，而 Methanosarcina 和 Methanothermobacter 组在 epPCR 应用中表现出最具问题的交叉反应。我们能够证明，用于检测总古菌群落、产甲烷目 Methanosarcinales、Methanobacteriales、Methanococcales 和 Methanoculleus 属的引物具有高度特异性，并允许在不使用昂贵的 TaqMan 探针的情况下准确定量目标生物。然而，设计用于检测 Methanomicrobiales、Methanothermobacter、Methanosarcina 和 Methanosaeta 的引物不适用于 SYBR Green 应用。通过变性梯度凝胶带定量和测序，评估了从高温发酵器获得的典型产甲烷群落的原位定量的可靠性。由此，我们揭示了产甲烷古菌的高丰度，主要由 Methanoculleus 和 Methanosarcinales 组成，而 Methanobacteriales 仅形成一小部分。
