16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Journal of Microbiological Methods
Timo NieminenPeter Neubauer

Abstract

Boreal soils have been suspected reservoirs of infectious environmental mycobacteria. Detection of these bacteria in the environment is hampered by their slow growth. We applied a quantitative sandwich hybridization approach for direct detection of mycobacterial 16S rRNA in soil without a nucleic acid amplification step. The numbers of mycobacterial 16S rRNA molecules found in the soil indicated the presence of up to 10(7) to 10(8) mycobacterial cells per gram of soil. These numbers exceed by factor of 10 to 100 x the previous estimates of mycobacteria in soil based on culture methods. When real-time PCR with mycobacteria targeting primers was used to estimate the number of 16S rDNA copies in soil, one copy of 16S rDNA was detected per 10(4) copies of 16S rRNA. This is close to the number of 16S rRNA molecules detected per cell by the same method in laboratory pure cultures of M. chlorophenolicum. Therefore a major part of the mycobacterial DNA in the studied soils may thus have represented metabolically active cells. The 16S rRNA sandwich hybridization method described in this paper offers a culture independent solution for tracking environmental reservoirs of viable and potentially infectious mycobacteria.

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Citations

Mar 23, 2013·Acta Veterinaria Scandinavica·Taneli TirkkonenJaakko Pakarinen
May 4, 2010·Journal of Microbiological Methods·Eila TorvinenAino Nevalainen Helena Rintala
Dec 13, 2006·Journal of Microbiological Methods·Sanna HuhtamellaPeter Neubauer
Aug 1, 2010·Environmental Microbiology Reports·Janne J KoskimäkiAnna Maria Pirttilä
Mar 22, 2014·Frontiers in Microbiology·Ekaterina OsmekhinaPeter Neubauer
Mar 8, 2019·Microbiology·Shravanthi S Kumar, Asit Ranjan Ghosh
Jun 30, 2019·Applied and Environmental Microbiology·Corinne M WalshNoah Fierer

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