[A digital droplet PCR detection technique based on filter faster R-CNN].

ABSTRACT

OBJECTIVE: To propose a method for mitigate the impact of anomaly points (such as dust, bubbles, scratches on the chip surface, and minor indentations) in images on the results of digital droplet PCR (ddPCR) detection to achieve high-throughput, stable, and accurate detection. METHODS: We propose a Filter Faster R-CNN ddPCR detection model, which employs Faster R-CNN to generate droplet prediction boxes followed by removing the anomalies within the positive droplet prediction boxes using an outlier filtering module (Filter). Using a plasmid carrying a norovirus fragment as the template, we established a ddPCR dataset for model training (2462 instances, 78.56%) and testing (672 instances, 21.44%). Ablation experiments were performed to test the effectiveness of 3 filtering branches of the Filter for anomaly removal on the validation dataset. Comparative experiments with other ddPCR droplet detection models and absolute quantification experiments of ddPCR were conducted to assess the performance of the Filter Faster R-CNN model. RESULTS: In low-dust and dusty environments, the Filter Faster R-CNN model achieved detection accuracies of 98.23% and 88.35% for positive droplets, respectively, with composite F1 scores reaching 99.15% and 99.14%, obviously superior to the other models. The introduction of the filtering module significantly enhanced the positive accuracy of the model in dusty environments. In the absolute quantification experiments, a regression line was plotted using the results from commercial flow cytometry equipment as the standard concentration. The results show a regression line slope of 1.0005, an intercept of -0.025, and a determination coefficient of 0.9997, indicating high consistency between the two results. CONCLUSION: The ddPCR detection technique using the Filter Faster R-CNN model provides a robust detection method for ddPCR under various environmental conditions.