A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

22 Here we report a novel microdroplet PCR method combined with fluorescence 23 spectrophotometry (MPFS), which allows for qualitative, quantitative and 24 high-throughput detection of multiple DNA targets. In this study, each pair of primers 25 was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA 26 was amplified and labeled with the same fluorophore. After products purification, the 27 DNA products tagged with different fluorophores could be analyzed qualitatively by 28 the fluorescent intensity determination. The relative fluorensence unit was also 29 measured to construct the standard curve and to achieve quantitative analysis. In a 30 reaction, the co-amplified products with different fluorophores could be 31 simultaneously analyzed to achieve high-throughput detection. We used four kinds of 32 GM maize as a model to confirm this theory. The qualitative results revealed high 33 specificity and sensitivity of 0.5% (w/w). The quantitative results revealed that the 34 limit of detection was 10 3 copies and with good repeatability. Moreover, 35 reproducibility assay were further performed using four foodborne pathogenic 36 bacteria. Consequently, the same qualitative, quantitative and high-throughput results 37 were confirmed as the four GM maize. 38