Elliptical Pipette Generated Large Microdroplets for POC Visual ddPCR Quantification of Low Viral Load.
Anal Chem
; 93(16): 6456-6462, 2021 04 27.
Article
em En
| MEDLINE
| ID: mdl-33861566
ABSTRACT
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 â¼150 to 350 µm in size with a CV of â¼2.3%. 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 µL 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sistemas Automatizados de Assistência Junto ao Leito
/
COVID-19
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos