Increased sensitivity using real-time dPCR for detection of SARS-CoV-2

METHOD SUMMARY A real-time dPCR instrument and assay was developed to improve detection limits and linear dynamic range of the assay, creating a more versatile and robust system for quantitating and detecting target nucleic acid sequences for infectious disease or other applications. The real-time data produced were analyzed by a simple set of heuristic functions to eliminate aberrant amplification profiles, resulting in a lower limit of detection. In addition, the fluorescent data from the entire chip can be averaged for each cycle during the amplification and used to generate a real-time amplification profile that provides quantitative information about the samples. A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. We compared three technologies - real-time qPCR, end point dPCR and real-time dPCR - in the context of SARS-CoV-2. Some improvement in limit of detection was obtained with end point dPCR compared with real-time qPCR, and the limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals. Real-time dPCR showed increased linear dynamic range compared with end point dPCR based on quantitation from amplification curves. Real-time dPCR can improve the performance of TaqMan assays beyond real-time qPCR and end point dPCR with better sensitivity and specificity, absolute quantification and a wider linear range of detection.

Automated summary

A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. The limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals, showing a larger linear dynamic range compared to end point dPCR.