Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

Instead of monitoring only the end-point fluorescent intensity, which is done in many single DNA molecular application experiments, our real-time fluorescence detector is useful for investigating amplification uniformity and for optimizing the total time required for incubation [74] . As shown in Fig 4, the RPA amplification is rapid, such that the number of positive points no longer increases after 15 min. Overall dRPA-on-chip processing requires less than 30 min, which is a 4-fold decrease compared to dPCR, with a processing time of approximately 2 h. The reduced time to results is especially important for fast field analysis and critical clinical applications, such as in cases of sepsis. Furthermore, real-time analysis improves detection sensitivity because it allows the use of a reference frame to subtract background noise, which cannot be achieved with a single end-point frame. Temporal information also helps to differentiate between amplified signals and noise because the rate at which the intensity changes can be expected to increase gradually from frame to frame, providing another parameter by which to detect positive droplets. A plot of fluorescence intensity vs. reaction time is shown in S3 Fig. We characterized the performance of the PWA chip for digital RPA using serial dilutions of a L. monocytogenes gDNA stock solution (Fig 5) . The expected concentration of the gDNA template was estimated as the number of copies per well (cpw), and the concentration of the original gDNA stock solution was verified spectrophotometrically. The detailed method for calculating the expected cpw is presented in the Preparation of dRPA reagents section. As the gDNA template was diluted, the expected cpw values were 9 × 10 -1 , 1.8 × 10 -1 , 3.6 × 10 -2 , 1.2 × 10 -2 , and 4 × 10 -3 . After incubation, the number of positive RPA wells on the PWA chip decreased proportionally (Fig 5a-5e) . No evidence of contamination was observed as no false positives were observed in the negative control (Fig 5f) . We repeated the experiments three times at each gDNA concentration to test the robustness and reproducibility of digital RPA on the PWA chip. A Poisson statistical analysis of the dRPA results was performed as previously described [35, 66] . As shown in Fig 6, the measured cpw with dRPA was highly concordant with the expected cpw, with an average error rate of less than 11% (N = 15). Accurate qPCR experiments can estimate original DNA concentrations with variation of less than 1 cycle number, resulting in 150-200% errors in original concentration estimates [75] ; accordingly, this dRPA method had better performance. Furthermore, it is noteworthy that potential pipetting errors during dilution and potential losses of DNA during sample preparation could increase the deviation between two estimates.