基于具有交叉反应活性探针的痕量核酸定量

An Absolute Quantitation of Minute DNA Templates by Cross-reactive Probes

Digital PCR (dPCR) offers several advantages over traditional real-time quantitative PCR. As dPCR relies on amplification from a single template by end point fluorescence, the Minimum Information for the Publication of Digital PCR Experiments (dMIQE) recommends that dPCR needs much deeper optimization. However, few current dPCR publications followed the recommendations. Here we used probes with cross-reactivity and performed droplet digital PCR (ddPCR), thus providing a strategy for dPCR optimization following the dMIQE guide. In this study, ddPCR was performed to quantify the rare sequence detection with cross-reactivity probes. The G or T allelic gene fragments of CCAT2 served as the template to mimic the minute sequence and the gradient dilution of allelic fragment templates were added to demonstrate the nonspecific amplitude as reference controls. The probe for rs6983267 from ThermoFisher showed cross-reactivity. Through temperature gradients optimization, 59℃ was selected for subsequent experiments. By performing ddPCR with the setting for nonspecific amplification directed by gradient templates, we quantified the minute sequence under the different settings especially for the nonspecific amplification (the concentration of T-allelic fragments is 6.8 copies/μL; the concentration of G-allelic fragments is 4.2 copies/μL; total concentration is 11 copies/μL). Importantly, the concentration results of the minute template involved was confirmed by high and low fluorescence signal from different channels. The strategy we described here partly fulfills the dMIQE recommendation and offers more confidence of ddPCR.