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-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.
Weller
Simon A
SA
Central Science Laboratory, DEFRA, Sand Hutton, York, YO41 1LZ, UK. s.weller@csl.gov.uk
Simpkins
Sean A
SA
Stead
David E
DE
Kurdziel
Andrew
A
Hird
Heather
H
Weekes
Rebecca J
RJ
D029688:Brassica napus / Q000382:microbiology*; D005456:Fluorescent Dyes; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012231:Rhizobium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012639:Seeds / Q000382:microbiology*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
D005456:Fluorescent Dyes; D019914:Taq Polymerase