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) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt-2 (pWW0), 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 dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR 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 fragments were designed to permit simultaneous amplification and detection 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. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 mu L) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits far the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of = 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.