(498e) Elliptical Pipette Generated Droplets for Digital PCR Quantification of Saliva Viral Load

Affordable accurate quantification of low viral load specimens is highly desired to significantly help contain a fast-spreading pandemic. Herein, we report a droplet digital PCR (ddPCR) platform without bulky lab-bound optical equipment specifically for this purpose. For Point-of-Care applications, these droplets must be detectable by eye or by a smart-phone camera. To allow viral load quantification by counting only the positive droplets, the molecules must be digitally distributed in the emulsion, with either one or no target molecule in each droplet. Consequently, the droplets must be uniform in size.

Monodispersed aqueous droplets about 150 to 400 microns in diameter are generated through oil-wetted flattened pipette tips with elliptical cross-sections. The elliptic geometry favors the azimuthal curvature such that recoil by the axial tension is not sufficient to generate satellite droplets. The rapid pinchoff also prevents coalescence in the classical dripping mode in commercial droplet generation technologies with flow focusing. An ellipse aspect ratio larger than a critical value of 3.5 is predicted from a lubrication hydrodynamic theory to produce sufficient asymmetry for the production of monodispersed droplets. This prediction is verified experimentally with droplet emulsions whose coefficient of variation is less than 3%. The enhanced Rayleigh-Plateau capillary instability also yields droplets whose size and distribution are insensitive to the flow rate, such that they can be generated manually with a hand operated pipette.

We are able to rapidly (<5 min) generate several thousand monodispersed large microdroplets containing the PCR mix and saliva sample in a PCR tube with fluorinated oil. The thermal stability of these large droplets in oil are further stabilized by a block copolymer surfactant (polyoxyalkylene F127) in aqueous reaction phase. The emulsion is thermal cycled in a portable thermal cycler and the emulsion is spread over a ring reservoir on a glass slide. Due to the large droplet size, fluorescence of the PCR molecular beacons in positive droplets can be detected visually and imaged by a smartphone with appropriate illumination/filtering. Several thousand droplets from each 20-µL PCR reaction statistically allow quantification up to 100 target copies simply by counting positive droplets in the Poisson limit. The ddPCR platform has been verified by 2019 nCoV-PCR assay to demonstrate a limit-of-detection of 3.8 copies per 20-µL reaction and a dynamic range of 4 to 100 copies. With some dilution, our ddPCR platform is demonstrated to be inhibitor resistant as shown by spiked saliva samples, implying RNA extraction is not necessary. Therefore, the ddPCR platform represents a rapid 1.5-hour inexpensive quantitative PCR test that can be easily set up even in resource-limited conditions with elliptical pipette droplet generation and smartphone camera imaging.