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Titolo:
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
Autore:
VANELSAS JD; MANTYNEN V; WOLTERS AC;
Indirizzi:
RES INST PLANT PROTECT,12 BINNENHAVEN,POB 9060 NL-6700 GW WAGENINGEN NETHERLANDS
Titolo Testata:
Biology and fertility of soils
fascicolo: 2, volume: 24, anno: 1997,
pagine: 188 - 195
SICI:
0178-2762(1997)24:2<188:SDEAAO>2.0.ZU;2-Z
Fonte:
ISI
Lingua:
ENG
Soggetto:
POLYMERASE CHAIN-REACTION; RHODOCOCCUS-CHLOROPHENOLICUS; MICROBIAL DNA; BACTERIA; PENTACHLOROPHENOL; MICROORGANISMS; SEDIMENTS;
Keywords:
SOIL DNA; 16S RIBOSOMAL RNA; MYCOBACTERIUM CHLOROPHENOLICUM; QUANTITATIVE PCR; IMMUNOFLUORESCENCE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
24
Recensione:
Indirizzi per estratti:
Citazione:
J.D. Vanelsas et al., "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", Biology and fertility of soils, 24(2), 1997, pp. 188-195

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 mu g 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.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/09/20 alle ore 07:48:59