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°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 102 cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples.
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Received 28 February 1997/ Accepted in revised form 23 November 1997
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Khan, A., Jones, R. & Cerniglia, C. Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments. J Ind Microbiol Biotech 20, 90–94 (1998). https://doi.org/10.1038/sj.jim.2900489
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DOI: https://doi.org/10.1038/sj.jim.2900489