Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR.
Anal Chem
; 93(9): 4208-4216, 2021 03 09.
Article
in English
| MEDLINE | ID: covidwho-1101611
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.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Multiplex Polymerase Chain Reaction
/
COVID-19 Testing
/
SARS-CoV-2
/
COVID-19
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
Anal Chem
Year:
2021
Document Type:
Article
Affiliation country:
Acs.analchem.0c04626
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