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Principal Investigator: Frederick R Haselton
Organization: VANDERBILT UNIVERSITY
Fiscal Year: 2024
Award: $762,178
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
The ongoing COVID-19/SARS-CoV-2 pandemic highlights the need for simple, rapid, and cost-effective
testing for respiratory infections at the point-of-care, including physician’s offices, urgent-care settings,
ambulatory procedural centers, and low-resource environments. The need is particularly notable for respiratory
infections, such as COVID-19 and influenza, which can present with similar symptoms yet require distinct
management strategies. With its high sensitivity and specificity, RT-PCR is the gold standard for the molecular
diagnosis and differentiation among respiratory pathogens. Traditional RT-PCR workflow requires significant
control over specimen contents and reaction conditions, with current methods requiring nucleic acid extraction
prior to amplification and detection. The net result is increased complexity, cost, and/or turnaround time for
diagnosis. In this context, we have observed in recent influenza studies that outstanding analytic performance
characteristics can be achieved without RNA extraction, by applying our novel workflow and Adaptive PCR
technology. Unlike traditional RT-PCR, Adaptive RT-PCR incorporates mirror-image L-DNA enantiomers—
identical in sequence to PCR primers and targets—that modify cycling conditions to match the biochemical
sample contents, thus eliminating the need to monitor reaction temperature. The direct monitoring of reaction
conditions overcomes many of the limitations of traditional PCR, facilitating direct amplification within the original
specimen matrix, simplifying instrument design, and enabling single-tube analyses.
SARS-CoV-2 and influenza are both enveloped RNA viruses, with specimens collected in the same
manner (i.e. nasopharyngeal swab) and using the same viral transport medium. Therefore, we hypothesize that
we may eliminate RNA extraction for this virus, like we have done for influenza, by performing Adaptive RT-PCR
directly on clinical specimens. We propose to enable a simplified methodology through Adaptive RT-PCR,
creating diagnostics for COVID-19 and other respiratory pathogens without RNA extraction. As a collaboration
between biomedical engineers and a COVID-19 diagnostic laboratory, we seek to develop a workflow and
instrument that are simple-to-use, cost-effective, and suitable for point-of-care settings, tools that can rapidly
inform treatment and management strategies. To achieve this goal, Aim 1 will evaluate the performance of RT-
PCR directly – that is, without RNA extraction – using both traditional and Adaptive RT-PCR instrumentation.
Aim 2 will develop multiplexed amplification reagents to create a sensitive and specific respiratory panel that
detects SARS-CoV-2, four other viruses, two bacteria, and one control target. Ultimately, Aim 3 will design and
fabricate a self-contained Adaptive RT-PCR instrument suitable for point-of-care settings, while validating this
system using characterized human specimens in a CLIA-accredited lab environment. Completion of this project
will result in a novel point-of-care tool for both the established and emerging respiratory infections that threaten
public health, facilitating rapid treatment, follow-up, infection prevention, and epidemiologic containment.
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