Event-Specific Qualitative and Quantitative Detection of Genetically Modified Rice G6H1
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Abstract
The genetically modified rice G6H1 expressing a fused protein of Cry1Ab/Vip3H and a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) G6 is a genetically modified event that has been approved for preproduction field testing in China. The purpose of this study was to establish an event-specific qualitative and quantitative detection method that could provide a stable, reliable system for monitoring this new transgenic event. In this study, event-specific qualitative and quantitative detection methods based on the 3′ integration junction sequence between host plant DNA and the integrated gene were developed. The limit of detection (LOD) of qualitative PCR was assessed to be 0.1%. The LOD of quantitative PCR was estimated to be ten haploid genome copies. The quantitative PCR detection method was verified with three mixed rice samples with known G6H1 contents, and the results agreed with the expected values. Evaluation of specificity and sensitivity indicated that the developed qualitative and quantitative PCR methods are reliable and can be used for the detection and quantification of G6H1.
Keywords
Genetically modified organism G6H1 rice Event-specific method Real-time PCRNotes
Acknowledgements
We greatly thank Shen Zhicheng of Zhejiang University for providing the seeds of the G6H1 event and non-transgenic rice Xiushui 110.
Funding
This work was financially supported by the National Transgenic Plant Special Fund (2014ZX08012-001) and a grant from Public Technology Application Research of Zhejiang Province (LGN18C200029).
Compliance with Ethical Standards
Conflict of Interest
Xiaoli Xu declares that she has no conflict of interest. Xiaoyun Chen declares that she has no conflict of interest. Yongmin Lai declares that he has no conflict of interest. Qing Zhang declares that he has no conflict of interest. Yuhua Wu declares that she has no conflict of interest. Jun Li declares that he has no conflict of interest. Cheng Peng declares that he has no conflict of interest. Xiaofu Wang declares that he has no conflict of interest. Qingmei Miao declares that she has no conflict of interest. Hui Liu declares that she has no conflict of interest. Junfeng Xu declares that he has no conflict of interest.
Ethical Approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed Consent
Not applicable
Supplementary material
References
- Akiyama H, Sakata K, Kondo K, Tanaka A, Liu MS, Oguchi T, et al (2008) Individual detection of genetically modified maize varieties in non-identity-preserved maize samples. J Agric Food Chem 56:1977–1983CrossRefGoogle Scholar
- Arumuganathan K, Earle ED (1991) Nuclear DNA content of some important plant species. Plant Mol Biol Report 9:208–218CrossRefGoogle Scholar
- Chen Y, Tian J, Shen ZC, Peng YF, Hu C, Guo YY, Ye GY (2010) Transgenic rice plants expressing a fused protein of Cry1Ab/Vip3H has resistance to rice stem borders under laboratory and field conditions. J Econ Entomol 103:1444–1453CrossRefGoogle Scholar
- Chen M, Shelton A, Ye GY (2011) Insect-resistant genetically modified rice in China: from research to commercialization. Annu Rev Entomol 56:81–101CrossRefGoogle Scholar
- ENGL (2015) Definition of minimum performance requirements for analytical methods of GMO testing. Available at: http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm
- Fraiture MA, Herman P, Taverniers I, Loose MD, Deforce D, Roosens NH (2013) An innovative and integrated approach based on DNA walking to identify unauthorized GMOs. Food Chem 147:60–69CrossRefGoogle Scholar
- Fraiture MA, Herman P, Taverniers I, Loose MD, Nieuwerburgh FV, Deforce D, Roosens NH (2014) Validation of a sensitive DNA walking strategy to characterize unauthorized GMOs using model food matrices mimicking common rice products. Food Chem 173:1259–1265CrossRefGoogle Scholar
- Hernandez M, Esteve T, Pla M (2005) Real-time polymerase chain reaction based assays for quantitative detection of barley, rice, sunflower, and wheat. J Agric Food Chem 53:7003–7009CrossRefGoogle Scholar
- Jensen AH, Bertheau Y, Loose MD, Grohmann L, Hamels S, Hougs L et al (2012) Detecting un-authorized genetically modified organisms (GMOs) and derived materials. Biotechnol Adv 30:1318–1335CrossRefGoogle Scholar
- Jiang L, Yang L, Zhang H, Guo J, Mazzara M, Van den Eede G et al (2009) International collaborative study of the endogenous reference gene, sucrose phosphate synthase (SPS), used for qualitative and quantitative analysis of genetically modified rice. J Agric Food Chem 57:3525–3532CrossRefGoogle Scholar
- Li Y, Hallerman EM, Liu Q, Wu K, Peng Y (2016) The development and status of Bt rice in China. Plant Biotechnol J 14:839–848Google Scholar
- Mäde D, Degner C, Grohmann L (2006) Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice. Eur Food Res Technol 224:271–278CrossRefGoogle Scholar
- Mazzara M, Grazioli E, Savini C, Van Den Eede G (2006) Event-specific method for the quantitation of rice line LLRICE62 using real-time PCR - validation report and protocol - sampling and DNA extraction of rice. EUR 22490 EN. JRC34091Google Scholar
- Ministry of Agriculture and Forestry of South Korea (2000) Guidelines for labeling of genetically modified agricultural products. MAF Notification 31Google Scholar
- Commission Regulation (EC) No. 1829 (2003) Of the European parliament and of the council of 22 September 2003 on geneticallymodified food and feed. Off J Eur Union L 268:1–23Google Scholar
- Yang Y, Li L, Yang H, Li X, Zhang X, Xu J, Zhang D, Jin W, Yang L (2018) Development of certified matrix-based reference material as calibrator for genetically modified rice G6H1 analysis. J Agric Food Chem 66:3708–3715CrossRefGoogle Scholar
- Zhao T, Lin CY, Shen ZC (2011) Development of transgenic glyphosate-resistant rice with G6 gene encoding 5-Enolpyruvylshikimate-3-Phophate synthase. Agric Sci China 10:1307–1312CrossRefGoogle Scholar