Use of quantitative real-time and conventional PCR to assess the stability of the cp4 epsps transgene from Roundup Ready canola in the intestinal, ruminal, and fecal contents of sheep.
The stability of transgenic DNA encoding the synthetic cp4 epsps protein in a diet containing Roundup Ready (RR) canola meal was determined in duodenal fluid (DF) batch cultures from sheep. A real-time TaqMan PCR assay was designed to quantify the degradation of cp4 epsps DNA during incubation in DF at pH 5 or 7. The copy number of cp4 epsps DNA in the diet declined more rapidly (P < 0.05) in DF at pH 5 as compared to pH 7. The decrease was attributed mainly to microbial activity at pH 7 and perhaps to plant endogenous enzymes at pH 5. The 62-bp fragment of cp4 epsps DNA detected by real-time PCR reached a maximum of approximately 1600 copies in the aqueous phase of DF at pH 7, whereas less than 20 copies were detected during incubations in DF at pH 5. A 1363-bp sequence of cp4 epsps DNA was never detected in the aqueous fraction of DF. Additionally, genomic DNA isolated from RR canola seed was used to test the persistence of fragments of free DNA in DF at pH 3.2, 5, and 7, as well as in ruminal fluid and feces. Primers spanning the cp4 epsps DNA coding region amplified sequences ranging in size from 300 to 1363 bp. Free transgenic DNA was least stable in DF at pH 7 where fragments less than 527 bp were detected for up to 2 min and fragments as large as 1363 bp were detected for 0.5 min. This study shows that digestion of plant material and release of transgenic DNA can occur in the ovine small intestine. However, free DNA is rapidly degraded at neutral pH in DF, thus reducing the likelihood that intact transgenic DNA would be available for absorption through the Peyer's Patches in the distal ileum.
Alexander
Trevor W
TW
Agriculture and Agri-Food Canada Research Centre, P.O. Box 3000, Lethbridge, Alta., Canada T1J 4B1.
Sharma
Ranjana
R
Deng
Ming Y
MY
Whetsell
Amy J
AJ
Jennings
James C
JC
Wang
Yuxi
Y
Okine
Erasmus
E
Damgaard
Dana
D
McAllister
Tim A
TA
D000818:Animals; D001483:Base Sequence; D001937:Brassica / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D005243:Feces / Q000737:chemistry*; D030841:Food, Genetically Modified; D042822:Genomic Instability / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives*; D007422:Intestines / Q000737:chemistry*; D008969:Molecular Sequence Data; D009862:Online Systems; D016133:Polymerase Chain Reaction / Q000379:methods*; D012417:Rumen / Q000737:chemistry*; D012756:Sheep; D000097797:Glyphosate
D018744:DNA, Plant; D005998:Glycine; D000097797:Glyphosate