Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Abstract

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.