EXTRACTION AND PURIFICATION OF MICROBIAL DNA FROM PETROLEUM-CONTAMINATED SOILS AND DETECTION OF LOW NUMBERS OF TOLUENE, OCTANE AND PESTICIDE DEGRADERS BY MULTIPLEX POLYMERASE CHAIN-REACTION AND SOUTHERN ANALYSIS

We investigated the use of multiplex polymerase chain reaction (PCR) t echniques coupled with Southern analysis to detect xenobiotic-degradin g organisms that had been added to three soils. Two soils highly conta minated with petroleum hydrocarbons and a less contaminated control so il were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW 0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissoluti on purification procedure. This DNA was subjected to multiplex polymer ase chain reaction (PCR) using primers that amplify regions of xylM (P CR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA f ragments were designed to permit simultaneous amplification and detect ion of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 10(0) to 10 (6) cells per gram soil, depending on the soil and the target gene. So uthern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and co nfirmed the identity of the PCR products. For some samples that were i nitially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibi tion of the PCR by diluting coextracted contaminants in the environmen tal DNA. A second PCR on an aliquot (1 mu L) of the first reaction inc reased the ethidium bromide-based detection limits for one of the soil s to six cells per gram of soil; it did not increase the detection lim its far the other soils. Therefore, the DNA extraction procedure and m ultiplex PCR permitted the simultaneous detection of three types of bi odegradative cells, at a lower detection limit of = 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitati ons of multiplex PCR, the amplified signals did not follow a close dos e response to the numbers of added target cells.