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Nuclemeter: A yardstick for quantifying nucleic acids undergoing enzymatic amplification

Point-of-care, reaction-diffusion column-based, non-instrumented device for real-time visual measurement of viral load in resource-limited settings, in the clinic, and at home

Problem:

Quantification of viral load in patients is needed to assess disease progression, monitor drug therapy efficacy, and identify the emergence of drug-resistant strains, particularly HIV. While real-time nucleic acid amplification and quantification methods, such as PCR, have revolutionized modern genetics and diagnostics, expensive specialized instrumentation and extensive sample preparation are needed, along with continuous monitoring of fluorescence from the DNA intercalating agent. Lateral flow strips can detect enzymatic amplification products, but at low sensitivity and provide only crude estimates of concentration.

Solution:

The Bau Lab has designed a low-cost, reaction diffusion-based instrument-free device to quantify target nucleotides. The device employs reverse transcription, isothermal enzymatic amplification.

The nuclemeter is comprised of a sample chamber and a reaction-diffusion conduit, which contains the reagents for enzymatic amplification and an intercalating dye fluorescent reporter. After the sample is introduced, the amplification reaction is thermally triggered. The amplified DNA migrates along the reaction-diffusion conduit, where the target concentration is deduced from the position of the reaction front, which propagates over time, in a manner akin to reading the temperature from a mercury thermometer. Additional columns can be added to the device to assay multiple targets from the same sample and to establish microbial flora or gene profile.

The device can be used at the point of care and at home.

Advantages:

Low-cost, straightforward measurement
Usable at point-of-care; instrument-free
Multiplex to assay multiple targets simultaneously
Compatible with high-throughput processing
Use in resource-limited regions without trained staff, laboratory facilities, or electricity

Applications:

Early stage quantitative screening for viruses or pathogens using DNA or RNA
Assess stage of disease progression through viral load and copy number quantification
Monitor drug therapy efficacy
Identify drug-resistant strains
Profile gut microbiome
Pathogen environmental detection

A photograph of a plastic chip housing four nuclemeters, on left, and fluorescence emission imaging from the nuclemeter used for HIV viral load testing at 32 minutes after incubation, on right. The sample chambers connected to reaction-diffusion conduits 1, 2, 3, and 4 contained 104, 103, 102, and 0 HIV-1 RNA templates.