Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol 71

Here we describe a quantitative PCR-based approach to estimating the relative abundances of major taxonomic groups of bacteria and fungi in soil. Primers were thoroughly tested for specificity, and the method was applied to three distinct soils. The technique provides a rapid and robust index of microbial community structure. The diversity of the bacterial and fungal communities in soil is extraordinary (8, 27, 29). High levels of bacterial and fungal diversity make quantifying and characterizing soil microbial communities a daunting task. In recent years, quantitative PCR (qPCR, also referred to as real-time PCR) has emerged as a promising tool for studying soil microbial communities (10, 12, 20, 28). qPCR is based on the real-time detection of a reporter molecule whose fluorescence increases as PCR prod-uct accumulates during each amplification cycle (23). The qPCR approach is somewhat unique among methods of com-munity analysis in that it allows for a relatively rapid yet quan-titative assessment of the abundances of specific phylogenetic groups of microorganisms in soil. In this paper, we describe a qPCR-based approach to as-sessing soil microbial community structure at broad taxonomic levels. We developed and tested nine individual qPCR assays to quantify the abundances of the dominant groups of bacteria and fungi found in the soil environment. qPCR assays were conducted in polypropylene 96-well plates on an ABI Prism 7000 sequence detection system (Ap-plied Biosystems). Each 25-l reaction contained the follow-ing: 12.5 l of ABsolute qPCR Master Mix (ABgene), 1.25 l of each primer (10 M; Invitrogen), 2.5 l bovine serum al-bumin (10 mg ml1; Promega), 1.0 l SYBRGreen dye (1,600