Abstract
Quantification of microbial functional genes enhances predictions of soil biogeochemical process rates, but reliance on low-throughput quantitative PCR (qPCR) limits the scope of ecological studies to a handful of targets. Here, we explore whether microfluidic qPCR (MFQPCR) is a viable high-throughput alternative for functional gene quantification, by evaluating the efficiency, specificity and sensitivity of 29 established and 12 newly designed primer pairs targeting taxonomic, nitrogen-cycling and hydrocarbon degradation genes in gDNA soil extracts, under three different sets of MFQPCR assay conditions. Without curation, commonly-used qPCR primer pairs yielded an extreme range of reaction efficiencies (25.9% - 100.1%), but when conditions were optimized, MFQPCR produced copy-number estimates comparable to traditional qPCR. To guide microbial soil ecologists considering adoption of MFQPCR, we present suggestions for primer selection, including omission of inosines, degeneracy scores of < 9, amplicon sizes of ≤ 211 bp, and GC content of 32-61%. We conclude that, while the nanoliter reaction volumes, rapid thermocycling and one-size-fits-all reaction conditions of MFQPCR necessitates more stringent primer selection criteria than is commonly applied in soil microbial ecology, the ability to quantify up to 96 targets in 96 samples makes MFQPCR a valuable tool for monitoring shifts in functional community abundances. MFQPCR will particularly suit studies targeting multiple clade-specific functional genes, or when primer design is informed by previous knowledge of the environment.
Highlights
Soil microbial communities perform a dazzling array of ecosystem services, from fixation of atmospheric nitrogen and the release of nutrients from rocks (Landeweert et al, 2001; Levy-Booth et al, 2014), to the degradation of organic pollutants and pesticides (Molina et al, 2009; Kumar et al, 2016)
To ensure microfluidic qPCR (MFQPCR) assays are of high-efficiency while capitalizing on the increased throughput that the platform affords, we suggest that selection of both primers and representative sequences for gBlock standards be informed by knowledge of which gene variants are present in the environment in question
MFQPCR is a valuable tool for quantifying microbial functional communities in soil, provided primer pair and assay conditions are stringently curated
Summary
Soil microbial communities perform a dazzling array of ecosystem services, from fixation of atmospheric nitrogen and the release of nutrients from rocks (Landeweert et al, 2001; Levy-Booth et al, 2014), to the degradation of organic pollutants and pesticides (Molina et al, 2009; Kumar et al, 2016). In recent years a number of alternatives to traditional qPCR have arisen which drastically improve throughput by either miniaturizing, automating, digitalizing or multiplexing reactions (Baker, 2010; Huggett et al, 2013). The most promising technologies that are addressing this gap include microarrays such as Geochip, which uses hybridization of an array of > 20,000 probes to semi-quantitate thousands of gene variants in a small number of samples (He et al, 2010); digital PCR, which uses sample partitioning into droplets or nanoliter chambers to detect individual copies of rare targets (Baker, 2012); and microfluidic qPCR (MFQPCR), which uses nanoliter reaction volumes and a system of pressurized valves and microfluidic channels to automate the mixing and thermocycling of up to 96 assays and 96 samples in a single chip (Spurgeon et al, 2008). MFQPCR has emerged as an attractive alternative, being cost-effective, customizable, and as it uses the same chemistry as traditional qPCR, theoretically produces directly comparable results (Miller et al, 2016)
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