Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology

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.