Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence |
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Authors: | J D van Elsas V Mäntynen A C Wolters |
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Institution: | (1) Research Institute for Plant Protection, 12 Binnenhaven, PO Box 9060, NL-6700 GW Wageningen, Netherlands, NL |
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Abstract: | Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain
reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain
PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency
was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial
cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix,
and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide
hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration.
Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches.
Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils,
and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence
cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils.
Received: 8 February 1996 |
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Keywords: | Soil DNA 16S ribosomal RNA Mycobacterium chlorophenolicum Quantitative PCR Immunofluorescence |
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