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Published March 9, 2021 | Supplemental Material + Published
Journal Article Open

Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR

Abstract

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4–6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR). HDPCR combines TaqMan chemistry and familiar workflows with robust encoding to enable far higher levels of multiplexing on a traditional qPCR system without an increase in cost or reduction in throughput. We utilize HDPCR with a custom 20-Plex assay, an 8-Plex assay using unmodified predesigned single-plex assays from Integrated DNA Technologies and a 9-Plex pathogen panel inclusive of SARS-CoV-2 and other common respiratory viruses. All three assays were successful when tested on a variety of samples, with overall sample accuracies of 98.8, 98.3, and 100%, respectively. The HDPCR technology enables the large install base of qPCR instrumentation to perform mid-density multiplex diagnostics without modification to instrumentation or workflow, meeting the urgent need for increased diagnostic yield at an affordable price without sacrificing assay performance.

Additional Information

© 2021 The Authors. Published by American Chemical Society. Under an Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Received: November 2, 2020; Accepted: February 11, 2021; Published: February 25, 2021. The authors thank Jona Cura, Le Ai Trinh, and Paul Yim of the Fraser Lab for their hospitality, help getting properly trained, and setup at the University of Southern California. Special thanks to Bryan Leatham, Richard Abanes, and Megan Allen of ChromaCode for providing oligos for the builds. The authors declare the following competing financial interest(s): L.J., D.Y., J.A, P.B., K.F., C.M. and A.R. are employed by ChromaCode Inc., a company that is commercializing HDPCR assays.

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Published - acs.analchem.0c04626.pdf

Supplemental Material - ac0c04626_si_001.zip

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Additional details

Created:
August 20, 2023
Modified:
October 23, 2023