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Applied and Environmental Microbiology, April 2001, p. 1613-1618, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1613-1618.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development and Application of a Most-Probable-Number-PCR Assay To Quantify Flagellate Populations in Soil Samples

Line Fredslund,1,2 Flemming Ekelund,2 Carsten Suhr Jacobsen,1,* and Kaare Johnsen1

Geological Survey of Denmark and Greenland, DK-2400 Copenhagen NV,1 and Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen, DK-2100 Copenhagen Ø,2 Denmark

Received 29 August 2000/Accepted 8 January 2001

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

* Corresponding author. Mailing address: Geological Survey of Denmark and Greenland, Department of Geochemistry, Thoravej 8, DK-2400 Copenhagen NV, Denmark. Phone: 45 38 14 20 00. Fax: 45 38 14 20 50. E-mail: csj{at}geus.dk.

Applied and Environmental Microbiology, April 2001, p. 1613-1618, Vol. 67, No. 4
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.4.1613-1618.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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