Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants

Abstract

Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella(formerly Zoogloea), Acinetobacter,Stenotrophomonas, Escherichia,Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.

Bacteria beneath the West Antarctic Ice Sheet

“…Our contention that the observed community is endogenous to the subglacial environment is supported by the extremely low diversity and the relationship of the observed community to previously characterized subglacial microbial communities (Foght et al, 2004;Skidmore et al, 2005;Nemergut et al, 2007). In addition, the observed community lacks commonly observed laboratory contaminants and no PCR products were observed in procedural controls (Tanner et al, 1998).…”

Identification of different bacterial DNAs in human coronary arteries

“…The finding of E. coli sequences from two coronary arteries was omitted, as they were owing to Taq DNA polymerase contamination with E. coli DNA detected from extraction control reagents [7]. These contaminants become well-represented in recombinant bacterial 16S rDNA clone libraries when studying low concentrations of bacterial DNA specimens [8,9]. All specimen extracts were positive by human β -actin PCR, indicating that the DNA extraction procedure was adequate and that inhibitors of the PCR reaction were not present.…”

Molecular methods for pathogen and microbial community detection and characterization: Current and potential application in diagnostic microbiology

“…Broad-range PCR assays should only be performed using good molecular laboratory practices as previously outlined (Mitchell et al, 2011). In addition, it is critical that a human DNA amplification control as well as a positive and negative control (Tanner et al, 1998;Millar et al, 2002;Bosshard et al, 2003b;Greub et al, 2005;Fenollar et al, 2008b) be included during sample extraction and carried throughout the procedure. Negative controls should consist of a negative sample control (i.e., an aliquot of a known negative clinical sample such as blood from a person without bacteraemia), and a negative reaction mixture control (i.e., contains all reagents but no clinical sample extract) in order to detect reagent contamination.…”