title: Detection of genetically modified microorganisms in soil using the most-probable-number method with multiplex PCR and DNA dot blot.
abstract: The principal objective of this study was to detect genetically modified microorganisms (GMMs) that might be accidentally released into the environment from laboratories. Two methods [plate counting and most-probable-number (MPN)] coupled with either multiplex PCR or DNA dot blots were compared using genetically modified Escherichia coli, Pseudomonas putida, and Acinetobacter oleivorans harboring an antibiotic-resistance gene with additional gfp and lacZ genes as markers. Alignments of sequences collected from databases using the Perl scripting language (Perl API) and from denaturing gradient gel electrophoresis analysis revealed that the gfp, lacZ and antibiotic-resistance genes (kanamycin, tetracycline, and ampicillin) in GMMs differed from the counterpart genes in many sequenced genomes and in soil DNA. Thus, specific multiplex PCR primer sets for detection of plasmid-based gfp and lacZ antibiotic-resistance genes could be generated. In the plate counting method, many antibiotic-resistant bacteria from a soil microcosm grew as colonies on antibiotic-containing agar plates. The multiplex PCR verification of randomly selected antibiotic-resistant colonies with specific primers proved ineffective. The MPN-multiplex PCR method and antibiotic-resistant phenotype could be successfully used to detect GMMs, although this method is quite laborious. The MPN-DNA dot blot method screened more cells at a time in a microtiter plate containing the corresponding antibiotics, and was shown to be a more efficient method for the detection of GMMs in soil using specific probes in terms of labor and accuracy.
authors.affiliation: Division of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, Republic of Korea.
authors.forename: Jinki
authors.initials: J
authors.lastname: Yeom
authors.forename: Yunho
authors.initials: Y
authors.lastname: Lee
authors.forename: Jaemin
authors.initials: J
authors.lastname: Noh
authors.forename: Jaejoon
authors.initials: J
authors.lastname: Jung
authors.forename: Jungsoon
authors.initials: J
authors.lastname: Park
authors.forename: Hyoju
authors.initials: H
authors.lastname: Seo
authors.forename: Jisun
authors.initials: J
authors.lastname: Kim
authors.forename: Jiwon
authors.initials: J
authors.lastname: Han
authors.forename: Che Ok
authors.initials: CO
authors.lastname: Jeon
authors.forename: Taesung
authors.initials: T
authors.lastname: Kim
authors.forename: Woojun
authors.initials: W
authors.lastname: Park
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D015139:Blotting, Southern / Q000379:methods*; D015169:Colony Count, Microbial / Q000379:methods*; D024881:Drug Resistance, Bacterial; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010802:Phylogeny; D012988:Soil Microbiology
chemical_list: D000900:Anti-Bacterial Agents