Counting soil microbial communities: The impact of qPCR platform and mastermix on accuracy and precision

Abstract

Quantitative polymerase chain reaction (qPCR) is widely used in soil microbial ecology to quantify microbial communities, but its accuracy can be compromised by coextracted inhibitors. Furthermore, large-scale international studies involving multiple laboratories or meta-analyses studies can introduce variation in qPCR results when data generated from different sources are compared. This study evaluated the performance of four commercial mastermixes across different soil types, a mock community, and a positive template control against three targets on three widely used platforms. Sensitivity to inhibitors was tested, with one mastermix affected, although this was mitigated by adding 1 mg/ml bovine serum albumin. Amplification success varied by mastermix, platform, gene, and sample matrix. Most mastermix-platform combinations showed low accuracy emphasizing the need for careful pairing. Precision was primarily influenced by gene target, followed by platform, sample matrix, and mastermix, and was reduced at lower template concentrations. Only 64.67% of intraassay (within an assay) measurements meet accepted thresholds. Interassay (between platforms) quantification was unreliable due to significant variability, which increased the risk of inaccurate data interpretation. The study highlights the necessity of considering inter- and intraassay variation, assay accuracy, and inhibitors that may impact sample amplification when utilizing qPCR for quantification of microbial communities in environmental samples.