Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments.
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.
Khan
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Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson AR 72079, USA.
Jones
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RA
Cerniglia
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CE
D050557:Catechol 2,3-Dioxygenase; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D049308:Dioxygenases; D005798:Genes, Bacterial; D005818:Genetic Engineering; D019015:Geologic Sediments / Q000382:microbiology; D010105:Oxygenases / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D016958:Pseudomonas putida / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D004269:DNA, Bacterial; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase