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Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model

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Abstract

Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25 % in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias −13.27 to 13.05 %). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

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Acknowledgments

This research was supported by grants from the Public Welfare Industry-Special Research Project of Ministry of Environmental Protection (201109028), the National Major Special Project for the Development of Transgenic Organisms (grant nos. 2014ZX08012-003, 2014ZX08012-002B, and 2014ZX08012-005), the Special Fund for Basic Scientific Research of Central Colleges, South-Central University for Nationalities (grant no. CZQ13009), and the Major Research Project of CAAS Science and Technology Innovation Program.

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Authors and Affiliations

  1. Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, 430062, Wuhan, People’s Republic of China

    Li Zhang, Yuhua Wu, Gang Wu, Yinglong Cao & Changming Lu

  2. Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Science, South-Central University for Nationalities, 182 Min-Yuan Road, 430074, Wuhan, China

    Li Zhang

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  1. Li Zhang
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  2. Yuhua Wu
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  3. Gang Wu
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  4. Yinglong Cao
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  5. Changming Lu
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Corresponding author

Correspondence to Changming Lu.

Additional information

Li Zhang and Yuhua Wu contributed equally to this work.

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Zhang, L., Wu, Y., Wu, G. et al. Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model. Anal Bioanal Chem 406, 6385–6397 (2014). https://doi.org/10.1007/s00216-014-8056-5

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  • DOI: https://doi.org/10.1007/s00216-014-8056-5

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