Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR

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.