POINT-OF-CARE AND IMPROVED DETECTION AND QUANTIFICATION OF BIOMOLECULES
Researchers at UCSF and the Chan Zuckerberg Biohub have developed a workflow for accurate bulk quantitation of droplet digital PCR, which can be used in point-of-care clinical settings with minimal sample processing.
The quantitation of nucleic acids is important for clinical applications, especially for detection of emergent pathogens like SARS-CoV-2. Quantitative PCR (qPCR) is the gold standard in many clinical settings but requires nucleic acid sample processing, hindering its usefulness in many point-of-care settings where rapid detection is critical. Further, qPCR provides only relative quantification, unless a standard curve is provided. In contrast, digital PCR affords several advantages over qPCR, including absolute quantification and increased accuracy at low copy number. Digital droplet PCR (ddPCR) employs microfluidics to partition samples for dPCR; while ddPCR is superior to qPCR, the requirement of microfluidic instrumentation often renders its adoption impractical in clinical settings.
Stage of Research
The inventors demonstrate accurate bulk quantitation of droplet digital PCR with common lab equipment and limited sample processing, obviating the need for microfluidics. The inventors demonstrate the method’s relevance to point-of-care diagnostics by quantifying SARS-CoV-2 viral load directly from patient nasopharyngeal samples, without nucleic acid isolation. They show that ddPCR, but not bulk qRT-PCR, accurately quantifies SARS-CoV-2 nucleic acids from patient crude lysate. Further, to eliminate the use of microfluidics and expand point-of-care utility, the inventors use emulsification by vortexing to partition the ddPCR samples. Different methods for bulk enumeration of positive droplets are validated using common equipment, including bulk colorimetric or fluorescence verification using portable Android or iPhone devices, gel electrophoresis, qPCR, and LAMP.
Applications
- Target nucleic acid quantification by combining benefits of ddPCR with simplicity, accessibility, and scalability of plate processing
- Accurate pathogen RNA quantification from crude lysate, without nucleic acid extraction and purification
Advantages
- Approach combines attributes of ddPCR (i.e., enhanced accuracy and robustness to inhibition) with plate-processing ability
- Digital droplet amplification improves viral load quantification by sequestering reaction inhibitors in separate droplets form target nucleic acid sequences
- Bulk emulsification by vortexing to perform ddPCR, and bulk quantitation produces superior results to qPCR alone and similar accuracy to microfluidic monodispersed ddPCR
- Analysis of polydispersed droplets (i.e., droplets of different size) generated by vortex emulsification increases assay dynamic
Stage of Development
Research – in vitro
Publications
Sun C, Liu L, Vasudevan HN, Chang K-C, Abate AR. Accurate bulk quantitation of droplet digital polymerase chain reaction. 2021. Anal Chem. DOI: 10.1021/acs.analchem.1c00877
Vasudevan HN, Xu P, Servellita V, Miller S, Liu L, Gopez A, Chiu CY, Abate AR. Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification. 2021. Scientific Reports. DOI: 10.1038/s41598-020-80715-1
Related Web Links
https://www.abatelab.org/
Keywords
Biomolecules, diagnosis, droplet digital PCR, fluorescence, high-throughput, microfluidics, SARS-CoV-2
Technology Reference
UCSF SF2020-227, Chan Zuckerberg CZB-173F
