QUANTITATION OF PSEUDOMONAS SP STRAIN B13(FR1) IN THE MARINE-ENVIRONMENT BY COMPETITIVE POLYMERASE CHAIN-REACTION

A method of competitive polymerase chain reaction (cPCR) was developed for quantitation of Pseudomonas sp. strain B13(FR1) released into the marine environment. Following DNA extraction and purification with CTAB (hexadecyltrimethyl ammonium bromide) from seawater inoculated with Pseudomonas sp. strain B13(FR1), a 712 bp fragment (B13-fragment) was co-amplified with a 588 bp internal standard. The internal standard had the same priming sequences as the B13-fragment and was added to calibration standards and samples at a constant concentration. The yield of the two cPCR products was measured on digitized images of Polaroid or X-ray films. The ratio of the two product yields from cPCR was used to make standard curves based on serially diluted DNA extracted from seawater inoculated with Pseudomonas sp. strain B13(FR1). When cPCR products were detected on ethidium bromide stained gels, the ratio of the two fragments was fog-linear from 400 cells per cPCR to 4 x 10(5) cells per cPCR. The log-linear range was extended to 4 cells per cPCR by hybridizing southern blots with a chemiluminescent probe. With the described method Pseudomonas sp. strain B13(FR1) was quantitated in seawater samples inoculated with 0.1 cells ml(-1) to 10(4) cells ml(-1).