Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

A - Papers appearing in refereed journals

Cullen, D. W., Nicholson, P. S., Mendum, T. A. and Hirsch, P. R. 1998. Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction. Journal of Applied Microbiology. 84 (6), pp. 1025-1034.

AuthorsCullen, D. W., Nicholson, P. S., Mendum, T. A. and Hirsch, P. R.
Abstract

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 by. 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 strains 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.

KeywordsBiotechnology & Applied Microbiology; Microbiology
Year of Publication1998
JournalJournal of Applied Microbiology
Journal citation84 (6), pp. 1025-1034
Digital Object Identifier (DOI)doi:10.1046/j.1365-2672.1998.00443.x
PubMed ID9717287
Open accessPublished as non-open access
Funder project or code224
444
Project: 031471
ISSN13645072
1364-5072
PublisherWiley

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