Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Abstract

To assess the effects of food processing on the detection and quantification of transgenic rice TT51-1 in processed food by polymerase chain reaction (PCR) technology, we monitored the presence of TT51-1 components in rice crackers at different processing stages using conventional PCR, quantitative real-time PCR (qPCR), and droplet digital PCR (ddPCR) with standard or validated primers and probes. In conventional PCR, relatively longer amplification targets, such as the Bacillus thuringiensis (Bt) gene (301 bp) and the event-specific target (274 bp), were barely detected in baked, fried or microwaved samples. In qPCR, the amplification fluorescence signal was detected in boiled, dried, baked, and microwaved samples, but barely observed in fried samples. Conventional PCR with the same primers used in qPCR detected the corresponding shorter targets in all samples. The conventional PCR results were mainly consistent with the results of qPCR. The results indicate that food processing directly affects the detection of transgenic components, and suggest that relatively shorter fragments should be selected as the amplification targets for this type of analysis. We established qPCR and duplex ddPCR methods for quantifying TT51-1. The results of an orthogonal experiment indicated that the optimal conditions for TT51-1/PLD duplex ddPCR were 500/250 nM of primers/probe combined with 58 °C annealing temperature. Both methods were feasible for quantitative detection of TT51-1 in processed samples, with duplex ddPCR being a more attractive method for detecting transgenic components in processed food due to its stability, accuracy, PCR inhibitor resistance, and the lack of a need for reference materials.