Cross-Reactivity of Prokaryotic 16S rDNA-Specific Primers to Eukaryotic DNA: Mistaken Microbial Community Profiling in Environmental Samples
16S ribosomal RNA gene sequences are characteristically used as gold-standard genetic marker for the determination of bacterial and/or archaeal biodiversity, and community profiling of environmental samples. The 16S rRNA amplicon analysis till-date is taken as a standard method for investigation and identification of uncultivable bacteria in microbial diversity studies. The accuracy of these analyses strongly depends upon the choice of primers. It is presumed that these primers do not participate in non-specific amplifications. In the present study, by in silico, PCR and denaturing gradient gel electrophoresis (DGGE) analysis, we have shown that primers do cross-react with eukaryotic DNAs as well, eventually leading to overestimation of microbial biodiversity. We further demonstrated that the overestimation is not only due to cross-reaction with eukaryotic mitochondrial or plastid DNA, but also with eukaryotic chromosomal DNA, that is ubiquitous in environmental samples. We tried to establish methanogenic diversity in municipal solid waste (MSW) leachates and cow dung samples before and after enrichment of the prokaryotic DNA from eukaryotic ones. Results revealed that bands disappeared/get lightened in bacterial 16S rRNA-based DGGE community profiles, after prokaryotic DNA enrichment, but not in mcrA-based community profiles.
KeywordsEnvironmental DNA/metagenome 16S rDNA universal primers mcrA Cross-reactivity with eukaryotic DNA DGGE
SSM is indebted to Jawaharlal Nehru University and PURSE program of Department of Science and Technology (DST) for funding. AK was supported by National Postdoctoral Fellowship funded by Science & Engineering Research Board (SERB), Govt. of India [Award No. PDF/2015/000474].
Compliance with Ethical Standards
Conflict of interest
The authors have no conflicting interests.
- 4.Ampe F, Miambi E (2000) Cluster analysis, richness and biodiversity indexes derived from denaturing gradient gel electrophoresis fingerprints of bacterial communities demonstrate that traditional maize fermentations are driven by the transformation process. Int J Food Microbiol 60:91–97. https://doi.org/10.1016/S0168-1605(00)00358-5 CrossRefGoogle Scholar
- 11.Dollive S (2013) Dynamics of microeukaryotes and archaea in the mammalian gut microbiome. University of Pennsylvania, PennsylvaniaGoogle Scholar
- 21.Huys G, Vanhoutte T, Joossens M et al (2008) Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities. Curr Microbiol 56:553–557. https://doi.org/10.1007/s00284-008-9122-z CrossRefPubMedGoogle Scholar
- 24.Lane D (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematic. Wiley, New York, pp 115–175Google Scholar
- 26.Maitra S, Kumar B, Kumar S et al (2015) Coss-reactivity of prokaryotic 16S rRNA gene-specific primers with genomes from eukaryotic organisms from marshlands. J Biol Nat 2:56–68Google Scholar
- 27.Mori H, Maruyama F, Kato H et al (2014) Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes. DNA Res 21:217–227. https://doi.org/10.1093/dnares/dst052 CrossRefPubMedGoogle Scholar
- 31.Pace N (1996) New perspective on the natural microbial world: molecular microbial ecology. ASM News 62:463–470Google Scholar
- 43.Yadav S, Kundu S, Ghosh SK, Maitra SS (2015) Molecular analysis of methanogen richness in landfill and marshland targeting 16S rDNA sequences. In: Archaea. https://www.hindawi.com/journals/archaea/2015/563414/. Accessed 19 Sep 2017