Microbial Ecology

, Volume 68, Issue 4, pp 834–841 | Cite as

How to Deal with PCR Contamination in Molecular Microbial Ecology

Methods

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.

References

  1. 1.
    Osborn AM, Smith CJ (2005) Molecular microbial ecology. Taylor & Francis, AbingdonGoogle Scholar
  2. 2.
    Van Dongen WFD, White J, Brandl HB et al (2013) Age-related differences in the cloacal microbiota of a wild bird species. BMC Ecol 13:11. doi:10.1186/1472-6785-13-11 PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Mühl H, Kochem A-J, Disqué C, Sakka SG (2010) Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood. Diagn Microbiol Infect Dis 66:41–9. doi:10.1016/j.diagmicrobio.2008.07.011 CrossRefPubMedGoogle Scholar
  4. 4.
    Spangler R, Goddard NL, Thaler DS (2009) Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria. PLoS One 4:e7010. doi:10.1371/journal.pone.0007010 PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Borst A, Box AT, Fluit AC (2004) False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy. Eur J Clin Microbiol Infect Dis 23:289–99. doi:10.1007/s10096-004-1100-1 CrossRefPubMedGoogle Scholar
  6. 6.
    Or A, Comay O, Gophna U (2013) In situ transplant analysis of free-living bacteria in a lotic ecosystem. Res Microbiol 164:262–9. doi:10.1016/j.resmic.2012.12.004 CrossRefPubMedGoogle Scholar
  7. 7.
    Biesbroek G, Sanders EAM, Roeselers G et al (2012) Deep sequencing analyses of low density microbial communities: working at the boundary of accurate microbiota detection. PLoS One 7:e32942. doi:10.1371/journal.pone.0032942 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Carroll NM, Adamson P, Okhravi N (1999) Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion. J Clin Microbiol 37:3402–4PubMedCentralPubMedGoogle Scholar
  9. 9.
    Champlot S, Berthelot C, Pruvost M et al (2010) An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS One. doi:10.1371/journal.pone.0013042 PubMedCentralPubMedGoogle Scholar
  10. 10.
    Corless CE, Guiver M, Borrow R, Fox AJ (2000) Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 38:1747PubMedCentralPubMedGoogle Scholar
  11. 11.
    Chang S-S, Hsu H-L, Cheng J-C, Tseng C-P (2011) An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents. PLoS One 6:e20303. doi:10.1371/journal.pone.0020303 PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Silkie SS, Tolcher MP, Nelson KL (2008) Reagent decontamination to eliminate false-positives in Escherichia coli qPCR. J Microbiol Methods 72:275–82. doi:10.1016/j.mimet.2007.12.011 CrossRefPubMedGoogle Scholar
  13. 13.
    Ranjard L, Poly F, Lata J et al (2001) Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability. Appl Environ Microbiol 67:4479–4487. doi:10.1128/AEM.67.10.4479 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Ecologie et Dynamique des Systèmes Anthropisés (FRE 3498)CNRS/Université de Picardie Jules VerneAmiensFrance
  2. 2.Department of ZoologyEdward Grey InstituteOxfordUK
  3. 3.Department of BiologyUniversity of BergenBergenNorway

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