Applied Microbiology and Biotechnology, Vol.47, No.6, 719-725, 1997
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 mu g 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.
Keywords:DIRECT DNA EXTRACTION;BACTERIAL-DNA;SEDIMENTS;AMPLIFICATION;PCR;HYBRIDIZATION;POPULATIONS;SEQUENCES;RECOVERY;CELLS