Cross-Reactivity of Prokaryotic 16S rDNA-Specific Primers to Eukaryotic DNA: Mistaken Microbial Community Profiling in Environmental Samples

• Published in: Current microbiology Vol. 75; no. 8; pp. 1038 - 1045
• Main Authors: Yadav, Shailendra, Kumar, Arvind, Gupta, Manish, Maitra, S. S.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.08.2018; Springer Nature B.V
• Subjects: Animals; Bacteria; Bacteria - classification; Bacteria - genetics; Bacteria - isolation & purification; Biodiversity; Biomedical and Life Sciences; Biotechnology; Cattle; Cattle manure; Communities; Cross-reaction; Cross-reactivity; Denaturing Gradient Gel Electrophoresis; Deoxyribonucleic acid; DNA; DNA fingerprinting; DNA Primers - genetics; DNA Restriction Enzymes - metabolism; DNA, Chloroplast - genetics; DNA, Mitochondrial - genetics; DNA, Ribosomal - genetics; Dung; Environment; Eukaryota - genetics; Feces - microbiology; Fungi - classification; Fungi - genetics; Fungi - isolation & purification; Gel electrophoresis; Gene Expression Profiling; Gene sequencing; Genetic markers; Leachates; Life Sciences; Microbial corrosion; Microbiology; Microorganisms; Mitochondrial DNA; Municipal solid waste; Municipal waste management; Polymerase Chain Reaction; Primers; Prokaryotes; Ribonucleic acid; RNA; RNA, Ribosomal, 16S - genetics; rRNA 16S; Solid waste management; 16S rDNA universal primers; Cross-reactivity with eukaryotic DNA; Environmental DNA/metagenome; DGGE; mcrA
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s00284-018-1482-4

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.