Abstract
This paper is the first part of a serial study investigating a quantification model freed from endogenous reference gene for genetically modified (GM) content by real-time polymerase chain reaction (PCR). The serial study involves two parts: (1) quantitative determination of GM DNA in haplo-species plant samples; (2) quantitative determination of GM DNA in multi-species plant samples. The paper describes a methodology to quantify the GM content in a DNA extract using on one hand real-time PCR to determine the amount of GM targets present and on the other hand absorbance reading at 260 nm to measure the total DNA present in sample. The ratio of both values is expressed as GM percentage. The most prominent dominance of the novel model is that the direct quantitative relation between the initial amount of target template in real-time PCR reaction (X 0) and the content of GM DNA in tested material (X SD) is established. Theoretical analysis indicates that the developed quantitative model relieved from the dependence on endogenous reference genes is suitable to quantify the GM content in haplo-species plant sample, in addition, it has the applicability in the quantitative detection of GM content in multi-species GM plant sample. A trail, in which 75 haplo-species GM plant samples were involved, was conducted to validate the suitability of the novel quantification model. The bias varied from 0.00 to 24.00% except a tested sample with lower level of GM content, and the precision expressed as coefficient of variation (CV) was from 2.81 to 25.00%. The limit of quantitation (LOQ) of the quantitative assay was as low as 0.1%. Compared with the previous papers and the performance requirements raised by European Network of GMO Laboratories (ENGL) for analytical methods of GMO testing, the results demonstrated that the established quantification model is a suitable alternative to the more traditional endogenous reference assay in the quantification of GM content in haplo-species plant sample.
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Abbreviations
- C D :
-
The DNA content in tested materials
- CE:
-
Capillary electrophoresis
- C t :
-
Cycle threshold
- CRMs:
-
Certified reference materials
- CV:
-
The coefficient of variation
- DNA:
-
Deoxyribonucleic acid
- dsDNA:
-
Double-strands DNA
- e :
-
The base number of natural logarithm
- E :
-
Efficiency of the PCR reaction
- E ex :
-
DNA extraction efficiency
- ELISA:
-
Enzyme linked immunosorbent assay
- ENGL:
-
European Network of GMO Laboratories
- EU:
-
European Union
- FAM:
-
6-Carboxy-fluorescein
- GC:
-
Gas chromatography
- GM:
-
Genetically modified
- GMO:
-
Genetically modified organism
- HPLC:
-
High performance liquid chromatography
- K :
-
A constant
- LOQ:
-
Limit of quantitation
- M g :
-
Genome molecular weight
- M gR :
-
The genome molecular weight of the species that GMO belongs to
- M gX :
-
The genomic molecular weight of GMO
- N C :
-
The copy number of target exogenous gene in GMO genome
- N CR :
-
The copy number of endogenous reference gene in genome
- N CX :
-
The copy number of exogenous target gene in genome
- OD260 :
-
Optical density at 260 nm
- OD320 :
-
Optical density at 320 nm
- p35S:
-
35S Promoter from Cauliflower Mosaic Virus
- PCR:
-
Polymerase chain reaction
- R 0 :
-
The initial amount of endogenous gene in PCR reaction
- R 2 :
-
Correlation coefficient
- R D :
-
The DNA content of total species
- RRS:
-
Roundup Ready™ soybean line GT 40-3-2
- R S :
-
The mass ratio of total ingredients of a certain species plant to the total ingredients of the tested materials
- RSD:
-
Relative standard deviation
- SD:
-
Standard deviation
- tNOS:
-
Terminator of nopaline synthase gene
- TAMRA:
-
6-Carbixytetramethylrhodamine
- V 1 :
-
The total volume of DNA extraction solution of tested materials
- V 2 :
-
The volume of DNA solution added in PCR reaction
- W :
-
The mass of tested materials
- X 0 :
-
The initial amount of target template in PCR reaction
- W T-DNA :
-
The mass of GM DNA in the serial PCR reaction
- W S-DNA :
-
The DNA mass of tested materials in PCR reaction
- X D :
-
The DNA content in GMO
- X S :
-
The mass ratio of total ingredients of GMO to the total ingredients of the tested materials
- X SD :
-
The mass% of GM DNA to the total DNA in tested materials
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Acknowledgments
This work was supported by Science and Technology Fundation of Guangdong Province (NO.2005B26001116); Natural Science Fundation of Guangdong Province (NO.06027682); and Shenzhen Bereau of Science Tecnology and Information [(2007) 334–339].
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Deng, P., Yang, D., Yang, Y. et al. A model freed from endogenous reference gene for quantification of genetically modified DNA by real-time PCR. 1. Quantification of DNA from genetically modified organisms in haplo-species materials. Eur Food Res Technol 227, 1485–1498 (2008). https://doi.org/10.1007/s00217-008-0871-5
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DOI: https://doi.org/10.1007/s00217-008-0871-5