Abstract:

Purpose: To determine the flanking sequence between exogenous and endogenous fragments using rapid
amplification of cDNA ends (5’-RACE) and consequently to examine genetically modified soybean (GMS) DAS-44406-
6. Methods: Specific primers and probes were designed based on the flanking sequences of exogenous fragments of DAS-
44406-6. The specificity of the developed method was validated by using it to detect a variety of other GM samples and non-
GM samples. DAS-44406-6 was used to prepare 6 content gradients to test the sensitivity of the method. At last, digital PCR
was applied to measure nucleic acid molecules for absolute quantification. Conclusions: A real-time PCR method has been
established for the identification of GMS soybean DAS-44406-6 with high specificity. The limit of detection (LODs) of the
real-time PCR method at template DNA concentration of 100 ng/reaction and 0.01% GM soybean content was 16.6 copies
of genomic DNA from DAS-44406-6. As for the LOD of the digital PCR method, the relative standard deviation (RSD) was
0.7% at template DNA concentration of 0.5 ng/reaction. The highly specific method combining real-time PCR and digital
PCR could meet the requirements for the detection of GMS DAS-44406-6.

Key words: genetically modified soybean, DAS-44406-6 event, event-specific detection, real-time PCR, digital PCR