Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load

Rapid point-of-care (POC) quantification of low virus RNA load would significantly reduce the turn-around time for the PCR test and help contain a fast-spreading epidemic. Herein, we report a droplet digital PCR (ddPCR) platform that can achieve this sensitivity and rapidity without bulky lab-bound equipment. The key technology is a flattened pipette tip with an elliptical cross-section, which extends a high aspect-ratio microfluidic chip design to pipette scale, for rapid (<5 min) generation of several thousand monodispersed droplets similar to 150 to 350 mu m in size with a CV of similar to 2.3%. A block copolymer surfactant (polyoxyalkylene F127) is used to stabilize these large droplets in oil during thermal cycling. At this droplet size and number, positive droplets can be counted by eye or imaged by a smartphone with appropriate illumination/filtering to accurately quantify up to 100 target copies. We demonstrate with 2019 nCoV-PCR assay LODs of 3.8 copies per 20 mu L of sample and a dynamic range of 4-100 copies. The ddPCR platform is shown to be inhibitor resistant with spiked saliva samples, suggesting RNA extraction may not be necessary. It represents a rapid 1.5-h POC quantitative PCR test that requires just a pipette equipped with elliptical pipette tip, a commercial portable thermal cycler, a smartphone, and a portable trans-illuminator, without bulky and expensive micropumps and optical detectors that prevent POC application.

Automated summary

The droplet digital PCR (ddPCR) platform reported in this study utilizes a unique flattened pipette tip design to achieve rapid, stable, and highly sensitive quantification of low virus RNA load without the need for bulky lab-bound equipment. The technology allows for fast generation of thousands of monodispersed droplets, which can be easily counted or imaged for accurate quantification of target copies. This platform shows promising potential for rapid virus detection and epidemic control, offering a convenient solution for point-of-care (POC) applications.