Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

Abstract

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity.

Contrasts between the cryoconite and ice-marginal bacterial communities of Svalbard glaciers

“…Schutte et al (2009). Although the detection of phylotypes comprising more than 0.1Á1% of the cumulative relative abundance of a microbial community by T-RFLP is recognized (Dunbar et al 2000;Courtney et al 2012), it cannot be assumed that T-RFLP can accurately resolve all phylotypes present in a given environmental sample, given the long-tail distribution of taxa evident in microbial communities (Bent & Forney 2008).…”

Arbuscular Mycorrhizal Fungi Community Structure, Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

“…The resulting PCR product was diluted 20 folds (using TE buffer) and used as a template for second round PCR with FLR3/FLR4, the size of the PCR product was approximately 380bp [ 34 ]. Courtney et al [ 35 ] proved that nested PCR amplification of AMF large subunit with FLR3/FLR4 followed by Alu I and Mbo I digestion was found to be effective in detecting AMF species present in very low frequency in the soil. FLR3/FLR4 was fluorescently labeled at 5’ end with FAM and HEX, respectively.…”

Impact Assessment of Pumping Parameters on Groundwater Bacterial Communities

“…In other words, if any variation of the BCS occurred between reference samples and water coming out of the pumping tubing as a consequence of the impact of lift, flow regime, or tubing material, it can be considered to be below the detection limit of the employed molecular fingerprinting technique. It has to be noted that the latter is enabling both a highly sensitive and a robust molecular diversity characterization of bacterial communities (Tiedje et al ; Thies ; Courtney et al ) and it was hence assumed to detect small variations in BCS. Modern molecular techniques for the characterization of microbial communities rely on DNA extraction and PCR amplification steps, which are sources of biases and inaccuracy (von Wintzingerode et al ; Delmont et al ).…”

“…Knowledge of biocenoses and more specifically of bacterial community structures (BCSs) in aquifers is still relatively limited and little is known about the identities and abundances of key bacterial species (Goldscheider et al ). At present, molecular biology provides highly sensitive and reproducible tools for the in‐depth characterization of BCS (Thies ; Courtney et al ; Ekblom and Galindo ). However, the power of these techniques contrasts with the scarce knowledge of the biases and variability introduced by sampling techniques.…”