Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction
Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction
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DOI：
10.1002/bit.20347
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发表时间：
2005-03-20
影响因子：
3.8
通讯作者：
Hwang, S
中科院分区：
文献类型：
--
作者：
Yu, Y;Lee, C;Hwang, S
实时聚合酶链反应（PCR）是一种高灵敏度的方法，可用于检测和定量微生物种群，而无需在厌氧过程和环境样品中培养它们。本研究旨在设计引物和探针，并利用TaqMan系统进行实时荧光PCR检测产甲烷菌。设计了六组特异性产甲烷引物和探针。这些集合分别检测四个目（甲烷球菌目、甲烷杆菌目、甲烷微生物目和甲烷菌目）以及甲烷菌目的两个科（甲烷菌科和甲烷菌科）沿着。我们还设计了通用引物和探针组，特异性地检测原核生物的16S rDNA和细菌和真菌的域，并且与TaqMan实时PCR系统完全兼容。通过测试从28个古细菌培养物中分离的DNA和分析潜在的假结果，以经验验证了每个引物和探针组的靶组特异性。一般来说，每种引物和探针组对靶组都具有很强的特异性。本研究中设计的引物和探针组可用于检测和定量厌氧生物过程和各种环境中的订单水平（在甲烷菌目的情况下为家族水平）产甲烷菌群。(C)2005 Wiley Periodicals，Inc.
Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments. (C) 2005 Wiley Periodicals, Inc.