Development and application of a most-probable-number-PCR assay to quantify flagellate populations in soil samples

This paper reports on the first successful molecular detection and quantifi cation of soil protozoa. Quantification of heterotrophic flagellates and na ked amoebae in soil has traditionally relied on dilution culturing techniqu es, followed by most-probable-number (MPN) calculations. Such methods are b iased by differences in the culturability of soil protozoa and are unable t o quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful dete ction of protozoa in soil by DNA techniques requires (i) the development an d validation of DNA extraction and quantification protocols and (ii) the co llection of sufficient sequence data to find specific protozoan 18S ribosom al DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using prime rs targeting a 700-bp sequence of the small-subunit rRNA gene. The method w as tested by use of gnotobiotic laboratory microcosms with sterile tar-cont aminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa popula tion estimates obtained by the PCR assay and a conventional MPN assay in th e three soils tested.