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 metho d involved the establishment of a protocol for extracting total DNA fr om soil with the least contamination, and the use of the polymerase ch ain reaction (PCR) to detect strain PCP-1 with primers targeted with P CP-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 f urther purified with phenol/chloroform extractions, ammonium acetate p recipitation and a G200 Sephadex gel-filtration column. DNA was extrac ted from soil supplemented with different concentrations of PCP-1 cell s. 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 pro cess several samples at the same time.