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Understanding Microbial Communities
Published in Jean F. Challacombe, Metabolic Pathway Engineering, 2021
All of the NGS sequencing technologies offer huge advantages for studies of microbial communities [76]. To characterize microbial communities using NGS technologies, two approaches are commonly used: amplicon and shotgun metagenome sequencing [77, 78]. Amplicon sequencing involves extracting the DNA from the cells in a microbial community, focusing on one or more marker genes that are conserved in many, if not all, organisms in the sample. PCR amplification of the target gene(s) results in DNA amplicons, which are sequenced and characterized. As already described above, 16S rRNA is often used as the marker gene for amplicon sequencing studies of prokaryotes, as it is highly conserved among bacterial species, and a good marker for characterizing phylogenetic relationships and to determine “Who is there?” and their relative abundances in a community [47, 79–82].
Quantitative PCR Approaches for Predicting Anaerobic Hydrocarbon Biodegradation
Published in Kenneth Wunch, Marko Stipaničev, Max Frenzel, Microbial Bioinformatics in the Oil and Gas Industry, 2021
Courtney R. A. Toth, Gurpreet Kharey, Lisa M. Gieg
Quantitative PCR is a well-established molecular method that allows for the simultaneous detection and quantification of target DNA. In conventional PCR, the amplified DNA product, also known as an amplicon, is detected in an end-point analysis. In qPCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle. Although the concept is relatively simple, there are specific issues in qPCR that developers and users of this technology should bear in mind.
A framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewater
Published in Critical Reviews in Environmental Science and Technology, 2022
Ishi Keenum, Krista Liguori, Jeanette Calarco, Benjamin C. Davis, Erin Milligan, Valerie J. Harwood, Amy Pruden
One emerging alternative to selecting one or a few ARGs to target is to instead apply a high throughput qPCR (HT-qPCR) array, which employs microfluidics or other commercial technology to simultaneously conduct multiple qPCR reactions in parallel (Ishii et al., 2013; Tourlousse et al., 2012; White et al., 2011; Zhu et al., 2013). HT-qPCR arrays can target at least 300 genes in one step (Sandberg et al., 2018) and have been applied to surface water, aquaculture, and wastewater samples (Ahmed et al., 2018; Bueno et al., 2019; Mathai et al., 2019; Quintela-Baluja et al., 2019). Sandberg et al. (2018) validated the efficacy of this approach in wastewater by targeting 39 ARGs simultaneously (48 genes overall). The findings aligned with past studies and traditional qPCR enumerations of ARGs in wastewater, but with 95% less time and materials. However, HT-qPCR has not been fully validated in terms of specificity (i.e., amplification of correct target) and is limited by lower sensitivity (i.e., higher detection limit) as a result of the small (nanoliter) reaction volumes (Stedtfeld et al., 2018; Waseem et al., 2019). Additional limitations to optimizing HT-qPCR for environmental monitoring stem from inability to simultaneously optimize annealing temperatures for multiple primers (Malhotra et al., 1998; Sipos et al., 2007). It is also not possible to collect the resulting amplicons and verify specificity via alternative means (e.g., DNA sequencing). The cost and need for specialized equipment is also a limiting factor for many labs. According to a Web of Science search, HT-qPCR is primarily used by a few research groups for ARG monitoring in the water environment (Figure S2, supplementary material). As commercial kits and laboratories enable HT-qPCR monitoring of ARGs, it is critical that they support reporting of traditional qPCR parameters and QA/QC criteria, such as the LOD/LOQ and efficiencies to support progress toward standardized ARG monitoring.