Event-Specific Quantitative Detection of Nine Genetically Modified Maizes Using One Novel Standard Reference Molecule

Abstract Despite rapid developments in the detection techniques for genetically modified organisms (GMOs), the event-specific PCR method with high specificity is still the most used technique. In this study, event-specific simplex and duplex qualitative and quantitative detection systems were developed targeting the 3 insertion site of GM maize SYN-E3272-5 (3272) construct. A reference molecule p3272 was constructed to act as positive control and as calibrator for quantitative analysis. The LOD for simplex and duplex qualitative PCR assays was 10 copies of p3272 control DNA. LOD and the LOQ for simplex and duplex quantitative PCR assays were 10 and 25 copies of p3272 DNA, respectively. Furthermore, four practical GM maize samples were quantified using the established simplex and duplex quantitative PCR systems by in-house validation. Results from five operators showed that the bias ranged from 3.44 to 17.24 in the simplex system and from 0.42 to 16.06 in the duplex system, respectively. These results demonstrated that the established event-specific simplex and duplex qualitative and quantitative PCR systems combined with the reference molecule p3272 are suitable for the detection of GM maize 3272 and its derived products.

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Qualitative and Quantitative PCR-Based Detection Methods for Authorized Genetically Modified Cotton Events in India

Journal of AOAC International ◽

10.5740/jaoacint.13-271 ◽

2014 ◽

Vol 97 (5) ◽

pp. 1299-1309 ◽

Cited By ~ 5

Author(s):

Rashmi Chhabra ◽

Gurinder Jit Randhawa ◽

Rajesh K Bhoge ◽

Monika Singh

Keyword(s):

Genetically Modified ◽

Cost Effective ◽

Detection Methods ◽

Quantitative Detection ◽

Bt Cotton ◽

Pcr Assay ◽

Genetically Modified Cotton ◽

Detection Assay ◽

Endogenous Reference ◽

The Cost

Abstract Qualitative diagnostics for all five commercialized genetically modified (GM) cotton events for insect resistance in India is being reported for the first time in this paper. The cost-effective and robust multiplex PCR (MPCR)-based detection assay, distinguishing the insect resistant transgenic Bt cotton events, viz., MON531, MON15985, Event 1, GFM-cry1A, and MLS-9124, has been developed. This decaplex PCR assay targets nine transgenic elements, viz., sequences of four transgenes, three transgene constructs, and two event-specific sequences along with one endogenous reference gene. The LOD of the qualitative MPCR assay was up to 0.1%. A quantitative detection method for four widely commercially cultivated GM cotton events, namely, MON531, MON15985, Event 1, and GFM-cry1A, covering 99.5% of the total area under GM cultivation in the country, is also reported. A construct-specific real-time PCR assay has been developed for quantification of these GM cotton events with LOQ <0.05% and LOD <0.025%. The developed assays will be of great use to screen for the presence/absence of authorized GM cotton events in unknown samples and to check the authenticity of GM cotton seed samples.

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