Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology
Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology
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DOI：
10.1038/sj.jim.2900546
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发表时间：
1998-09-01
影响因子：
3.4
通讯作者：
Chandler, DP
中科院分区：
文献类型：
--
作者：
Chandler, DP
PCR技术在环境和工业微生物学中的应用由于无数的有机和无机污染物以及与核酸共化的酶抑制剂而变得复杂。这些复杂情况在定量PCR （qPCR）方法中更为复杂，这些方法基于对扩增效率的微妙但重要的假设和样品相对于获得的环境或工业过程的代表性。在低生物量和/或低模板的情况下，与样品中靶基因空间异质性、差异DNA（或RNA）提取效率、分子采样误差、PCR抑制剂衰减和扩增偏差相关的其他问题可能会迅速破坏传统竞争性PCR （cPCR）和最可能数PCR （MPN-PCR）格式的基本假设。因此，在环境微生物学和低模板枚举的背景下，提出了对cPCR和MPN-PCR假设的关键评估。qPCR假设分析的基本结论是：(a)环境qPCR计数总是估计值，而不是绝对计数，这是相对于PCR标准的；(b)传统的cPCR测定法不适合环境应用，特别是在低生物量情况下；(c) cPCR和传统MPN-PCR方法都没有充分考虑到在PCR计数中出现并被放大的田间规模、工艺水平或实验变化。因此，与样本复制相关的样本代表性和错误往往比与qPCR测定本身相关的错误更重要。基于对qPCR假设的批评，描述了常规环境应用的替代qPCR方法，该方法基于复制限制稀释分析和分析灵敏度与实际效用之间的实用权衡。
The application of PCR techniques in environmental and industrial microbiology is complicated by innumerable organic and inorganic contaminants and enzyme inhibitors that copurify with nucleic acids. These complications are compounded in quantitative PCR (qPCR) methods, which are predicated upon subtle yet significant assumptions of amplification efficiency and the representativeness of the sample with respect to the environment or industrial process from which it was obtained. In low-biomass and/or low-template situations, additional concerns related to target gene spatial heterogeneity in the sample, differential DNA (or RNA) extraction efficiency, molecular sampling error, attenuation of PCR inhibitors and amplification bias can quickly undermine fundamental assumptions of conventional competitive PCR (cPCR) and most-probable-number PCR (MPN-PCR) formats. A critical evaluation of cPCR and MPN-PCR assumptions is therefore presented within the context of environmental microbiology and low-template enumerations. Fundamental conclusions from the analysis of qPCR assumptions are that: (a) environmental qPCR enumerations are invariably estimates, not absolute enumerations, which are relative to the PCR standard; (b) traditional cPCR assays are ill-suited for environmental applications, especially in low-biomass situations; and (c) both cPCR and traditional MPN-PCR practices insufficiently account for field-scale, process-level or experimental variations that arise and become amplified in PCR enumerations. Thus, sample representativeness and errors related to sample replication are frequently more important than errors related to the qPCR assay itself. Based upon this critique of qPCR assumptions, an alternate qPCR method for routine environmental application is described which is based upon replicative limiting dilution analysis and the pragmatic tradeoffs between analytical sensitivity and practical utility.