Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments

J Ind Microbiol Biotechnol. 1998 Feb;20(2):90-4. doi: 10.1038/sj.jim.2900489.

Abstract

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100 degrees C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10(2) cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples.

MeSH terms

  • Catechol 2,3-Dioxygenase
  • Cloning, Molecular
  • DNA, Bacterial / analysis
  • DNA, Bacterial / genetics
  • Dioxygenases*
  • Genes, Bacterial
  • Genetic Engineering*
  • Geologic Sediments / microbiology
  • Oxygenases / genetics
  • Plasmids
  • Polymerase Chain Reaction / methods*
  • Pseudomonas putida / genetics*
  • Pseudomonas putida / isolation & purification*
  • Sensitivity and Specificity
  • Soil Microbiology*

Substances

  • DNA, Bacterial
  • Oxygenases
  • Dioxygenases
  • Catechol 2,3-Dioxygenase