Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR: Implications for GM screening procedures

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-s...

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Bibliographic Details
Published in:	Archives of microbiology Vol. 178; no. 5; pp. 338 - 343
Main Authors:	WELLER, Simon A, SIMPKINS, Sean A, STEAD, David E, KURDZIEL, Andrew, HIRD, Heather, WEEKES, Rebecca J
Format:	Journal Article
Language:	English
Published:	Heidelberg Springer 01-11-2002 Berlin
Subjects:	Agronomy. Soil science and plant productions Biological and medical sciences Brassica napus - microbiology Fluorescent Dyes Fundamental and applied biological sciences. Psychology Genetics and breeding of economic plants Plasmids - genetics Polymerase Chain Reaction - methods Rhizobium - classification Rhizobium - genetics Rhizobium - isolation & purification Seeds - microbiology Seeds and other planting stocks Sensitivity and Specificity Taq Polymerase - metabolism Genetic marker Method Canola Contamination Polymerase chain reaction Agrobacterium Cruciferae Dicotyledones DNA Seed Angiospermae Brassica napus Bacteria Oil seed rape Spermatophyta Transgenic plant TaqMan PCR Rhizobiaceae Detection Genetically modified organism Oil plant (vegetal)
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Summary:	A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0302-8933 1432-072X
DOI:	10.1007/s00203-002-0461-z