Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction.
Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn5; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strain provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g-1 soil in soil-extracted DNA.
Cullen
D W
DW
Soil Science Department, IACR-Rothamsted, Harpenden, UK.
Nicholson
P S
PS
Mendum
T A
TA
Hirsch
P R
PR
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D015342:DNA Probes; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005818:Genetic Engineering; D016133:Polymerase Chain Reaction / Q000379:methods*; D012231:Rhizobium / Q000235:genetics*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D000900:Anti-Bacterial Agents; D015342:DNA Probes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial