How to Deal with PCR Contamination in Molecular Microbial Ecology

Abstract

Microbial ecology studies often use broad-range PCR primers to obtain community profiles. Contaminant microbial DNA present in PCR reagents may therefore be amplified together with template DNA, resulting in unrepeatable data which may be difficult to interpret, especially when template DNA is present at low levels. One possible decontamination method consists in pre-treating PCR mixes with restriction enzymes before heat-inactivating those enzymes prior to the start of the PCR. However, this method has given contrasting results, including a reduction in PCR sensitivity. In this study, we tested the efficiency of two different enzymes (DNase 1 and Sau3AI) as well as the effect of dithiothreitol (DTT), a strong reducing agent, in the decontamination procedure. Our results indicate that enzymatic treatment does reduce contamination levels. However, DNase 1 caused substantial reductions in the bacterial richness found in communities, which we interpret as a result of its incomplete inactivation by heat treatment. DTT did help maintain bacterial richness in mixes treated with DNase 1. No such issues arose when using Sau3AI, which therefore seems a more appropriate enzyme. In our study, four operational taxonomic units (OTU) decreased in frequency and relative abundance after treatment with Sau3AI and hence are likely to represent contaminant bacterial DNA. We found higher within-sample similarity in community structure after treatment with Sau3AI, probably better reflecting the initial bacterial communities. We argue that the presence of contaminant bacterial DNA may have consequences in the interpretation of ecological data, especially when using low levels of template DNA from highly diverse communities. We advocate the use of such decontaminating approaches as a standard procedure in microbial ecology.

Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR

“…However, a fully developed method will also require robust DNA extraction methods from large blood volumes but with small elution volumes, and ultrapure extraction reagents. The described method could also have direct utility in the field of microbial ecology, where PCR contaminants commonly complicate investigations [ 28 , 29 ]. In both settings, the long 16S rRNA gene amplicon designed here would provide considerable speciation power.…”

The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies

“…The amplification of background contaminants from PCR reagents could perhaps be avoided via the use of primer-extension PCR ( 102 ), but this would have no effect on contamination originating from other sources. Several methods have been suggested to remove contaminating DNA from reagents and the lab environment, including UV and gamma radiation ( 103 – 107 ); DNA intercalation by 8-methoxypsoralen, ethidium monoazide, and propidium monoazide ( 104 , 106 – 108 ); enzymatic treatments ( 105 – 107 , 109 – 111 ); silica-based membrane filtration ( 112 ); CsCl 2 density gradient centrifugation ( 111 ); and bleach/copper-bis-(phenanthroline)-sulfate/H 2 O 2 (CoPA) solution treatment ( 105 ). These methods have shown varied effects on contamination levels and PCR sensitivity, and the inclusion of reagent-only controls alongside these decontamination measures is still recommended.…”

A guide to minimize contamination issues in microbiome restoration studies

“…At this stage, samples are highly sensitive to contamination. As the concentration of unamplified DNA within samples is relatively low, any external or cross‐contamination will be amplified via PCR, and identifying cross‐contamination after this stage is impossible (Mennerat & Sheldon 2014; Walker 2019). Therefore, maintaining good pre‐PCR contamination control is extremely important.…”