4. Quantitative Polymerase Chain Reaction

4.1 Summary of qPCR

The quantitative polymerase chain reaction (qPCR)A laboratory analytical technique for quantification of a target gene based on DNA., also called quantitative real-time polymerase chain reactionMakes copies of a specific DNA sequence within a target gene of microorganisms that can be further analyzed., and reverse transcriptase-qPCR (RT-qPCR) methods are typically used to quantify the abundance and activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene) of target microorganisms capable of contaminant biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011). or of genes (DNA by PCR and qPCR) or transcripts (RNA by RT-qPCR) in biodegradation pathways. These methods can provide a direct line of evidence for evaluating the feasibility and performance of the biodegradation processes underlying monitored natural attenuation (MNA) and bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation. strategies. Traditionally, cultivation-based methods that rely on growth of the target microorganisms in the laboratory (such as microcosms, plate counts, or most probable number (MPN) analyses) have been used to estimate contaminant-degrading microbial populations. However, cultivation dependent techniques are laborious, time-consuming, and most importantly biased, because over 99% of microorganisms present in the environment cannot be grown under standard laboratory conditions (Amann et al. 1995).

Additional introductory information regarding PCR, qPCR, and RT-qPCR methods is available in the PCR Fact Sheet and the qPCR Fact Sheet. In addition, the qPCR Fact Sheet includes a list of current qPCR targets.

4.2 Applications

Site characterization, remediation, monitoring, and site closure require thorough examination of available chemical, geochemical, and microbiological data. In addition to identifying contaminants and delineating the spatial extent of contamination, site characterization includes defining baseline conditions for a preliminary evaluation of potential remedies (including MNA). The purpose of performing qPCR and RT-qPCR analyses is to provide the microbiological lines of evidence: quantification of the abundance and activity of microorganisms capable of biodegradation of the relevant contaminants at a site. Examples of sites where qPCR and RT-qPCR have been successfully used are presented in Table 4-1.

4.3 Data Interpretation

The application, analysis, and interpretation of microbiology-based EMD methods differ from typical soil and groundwater geochemical measurement in a number of ways. For example, microbial biomarkers cannot easily be preserved, and sample handling and processing requires special care. The analysis of microbial parameters requires specific data quality considerations for sampling plans, sample collection and handling, quality control and laboratory procedures, and these are discussed in Section 10. Included below is a brief introduction to how qPCR data are typically reported and some specific examples of how the data would be interpreted in answering the questions presented in Table 2-3.

4.3.1 How are the data typically reported?

4.3.2 How are qPCR data interpreted?

Interpretation of qPCR and RT-qPCR results depends on the contaminant, the biodegradation pathway, and site-specific factors including geochemical conditions and stage in the overall project life cycle. To illustrate interpretation of qPCR and RT-qPCR results, each of the questions relevant to qPCR and RT-qPCR in Table 2-3 is discussed below.

4.3.3 Practical considerations

The perceived limitation of DNA-based technologies, including qPCR, is that the detection of a target gene as an indicator of a specific microorganism or biodegradation pathway does not necessarily indicate corresponding activity. As discussed below, RT-qPCR is based on RNA rather than DNA, and is commercially available. RT-qPCR has been used to quantify gene transcription and microbial activity in environmental samples (Baldwin et al. 2010). Site managers can thus use qPCR as an indicator of biodegradation processes and RT-qPCR for confirmation of these processes.

4.4 Additional Information

Further reading specific to qPCR methods is provided in Appendix F.

Publication Date: April 2013

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