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Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples

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Abstract

Real time PCR assays were developed to detect and quantify the transgene DNA of a commercially released Bacillus thuringiensis (Bt) corn (Zea mays L.) hybrid (DKC42-23), which was derived from the event MON863 and also carried a neomycin phosphotransferase gene (the nptII gene). We applied the real time PCR assays to investigate the persistence of the transgene DNA in a field trial grown with DKC42-23 over 3 years, in combination with bacterial natural transformation. The results showed that under continuous cultivation of DKC42-23, its transgene DNA was detectable in the field plots all year around. Meanwhile, when soil DNA extracts from DKC42-23 plots were used as donor in bacterial natural transformation, successful recovery of kanamycin resistant (KmR) transformants indicated that the nptII gene carried by DKC42-23 could be taken up and integrated into naturally competent Pseudomonas stutzeri pMR7 cells, leading to the restoration of the antibiotic resistance of P. stutzeri pMR7. However, after the cultivation of a soybean line in the same plots for the subsequent growing season, the presence of transgene DNA of DKC42-23 was reduced to undetectable levels at the end of that growing season. Therefore, existing corn-soybean crop rotation practices reduce the availability of transgene DNA in soil and thus minimize the risks that might be attributable to horizontal gene transfer. The real time PCR assays are useful for investigating the persistence of transgene DNA derived from the MON863 event in soil environments.

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Acknowledgments

We would like to thank Dr. W. Wackernagel (University of Oldenburg, Germany) for providing P. stutzeri pMR7 and the reviewers for their thoughtful comments. This research was financially supported by the Canadian Environmental Protection Act (CEPA) and the Strategic Technology Applications of Genomics in the Environment (STAGE) Program.

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Correspondence to Bin Zhu.

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Zhu, B., Ma, B. & Blackshaw, R.E. Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples. Transgenic Res 19, 765–774 (2010). https://doi.org/10.1007/s11248-009-9353-1

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Keywords

  • Transgene DNA
  • Quantification
  • Genetically modified (GM)
  • Real time PCR
  • Soil environments
  • Bacillus thuringiensis (Bt)
  • Antibiotic resistance gene