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Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR

Jacky, Lucien and Yurk, Dominic and Alvarado, John and Belitz, Paul and Fathe, Kristin and MacDonald, Chris and Fraser, Scott and Rajagopal, Aditya (2021) Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR. Analytical Chemistry, 93 (9). pp. 4208-4216. ISSN 0003-2700.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription
Jacky, Lucien0000-0001-5570-7925
Yurk, Dominic0000-0002-2276-4189
Fraser, Scott0000-0002-5377-0223
Rajagopal, Aditya0000-0002-7768-2463
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.
Subject Keywords:Testing and assessment, Assays, Viruses, Genetics, Probes
Issue or Number:9
Record Number:CaltechAUTHORS:20210301-100548631
Persistent URL:
Official Citation:Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR. Lucien Jacky, Dominic Yurk, John Alvarado, Paul Belitz, Kristin Fathe, Chris MacDonald, Scott Fraser, and Aditya Rajagopal. Analytical Chemistry 2021 93 (9), 4208-4216; DOI: 10.1021/acs.analchem.0c04626
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108243
Deposited By: Tony Diaz
Deposited On:01 Mar 2021 19:22
Last Modified:12 Mar 2021 18:17

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