Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction.
A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 microgram soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time.
Lévesque
M J
MJ
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Laval, Québec, Canada.
La Boissière
S
S
Thomas
J C
JC
Beaudet
R
R
Villemur
R
R
D001421:Bacteria, Anaerobic / Q000302:isolation & purification*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S