Development and application of a most probable number-PCR assay to quantify flagellate populations in soil samples
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Development and application of a most probable number-PCR assay to quantify flagellate populations in soil samples. / Fredslund, Line; Ekelund, Flemming; Jacobsen, Carsten Suhr; Johnsen, Kaare Eske.
I: Applied and Environmental Microbiology, Bind 67, Nr. 4, 2001, s. 1613 - 1618.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Development and application of a most probable number-PCR assay to quantify flagellate populations in soil samples
AU - Fredslund, Line
AU - Ekelund, Flemming
AU - Jacobsen, Carsten Suhr
AU - Johnsen, Kaare Eske
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
U2 - 10.1128/AEM.67.4.1613-1618.2001
DO - 10.1128/AEM.67.4.1613-1618.2001
M3 - Journal article
C2 - 11282613
VL - 67
SP - 1613
EP - 1618
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
SN - 0099-2240
IS - 4
ER -
ID: 8673279