2016-809 Enhanced Fluorescence Readout and Reduced Inhibition for Nucleic Acid Amplification Tests

SUMMARY

UCLA researchers in the Department of Bioengineering have developed an enhanced fluorescent detection method for nucleic acid amplification tests.

BACKGROUND

Nucleic acid amplification tests are crucial elements in point-of-care diagnostics, such as for viral pathogens such as SARS-CoV-2. Isothermal methods that are easy to implement are especially well-suited for cost-effective testing. The use of fluorescent dyes and probes is a common component of many nucleic acid amplification tests as they can provide a readout for a variety of methods including PCR, NASBA, RCA, MDA, LAMP, Immuno-PCR, etc. Real-time fluorescence monitoring of amplification often requires costly custom probes, or fluorescent intercalating dyes, such as EvaGreen, Sybr Green, Pico green, and others. However, these dyes can often hinder the amplification process, making their use more challenging and time-consuming extending the reaction time. The development of a novel method or formulation preventing interference from the dyes would allow for improved real-time fluorescence monitoring, reduced cost optical readout systems, and lower the limit of detection benefiting the nucleic acid amplification testing field.

INNOVATION

Prof. Dino Di Carlo and colleagues at UCLA have developed a novel enhanced fluorescent readout formulation containing a dye quenching reagent that not only allows for >50-fold enhanced signal to background in real-time fluorescent monitoring of amplification from loop mediated isothermal amplification, as well as stability, leading to lower limits of detection, but also decreased time to readout. Their formulation containing EvaGreen and a dye sequestering reagent reduced readout time to 25-40- mins 2-fold for loop mediated isothermal amplification, roughly twice as fast as compared to using the dye alone. The technique has also been demonstrated in a single-molecule digital nucleic acid readout approach and in combination with ferrofluid based compartmentalized assays. This method allows for easy of integration of these dyes into current amplification techniques, improving performance with a simple change to the dye formulation without the need for costly custom probes.

POTENTIAL APPLICATIONS

• Early and rapid nucleic acid detection of bacterial or viral infection
• Point-of-care diagnostics device
• Isothermal detection of SAR-CoV-2 nucleic acids

ADVANTAGES

• Formulation is easily incorporated into many nucleic acid amplification techniques
• Significantly improved signal generation allowing for lower  limits of detection
• Real-time fluorescent readout for amplification analysis without costly reagents or readers
• Yields rapid results in 25-40 mins

RELATED MATERIALS

• Fractal LAMP: Label-Free Analysis of Fractal Precipitate for Digital Loop-Mediated Isothermal Nucleic Acid Amplification. Muñoz HE, Riche CT, Kong JE, van Zee M, Garner OB, Ozcan A, Di Carlo D. ACS Sens. 2020
• Ferrodrop Dose-Optimized Digital Quantification of Biomolecules in Low-Volume Samples. Kahkeshani S, Kong JE, Wei Q, Tseng D, Garner OB, Ozcan A, Di Carlo D. Anal Chem. 2018
• Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout. Kong JE, Wei Q, Tseng D, Zhang J, Pan E, Lewinski M, Garner OB, Ozcan A, Di Carlo D. ACS Nano 2017

STATUS OF DEVELOPMENT

Researchers have validated their novel formulation using LAMP (Loop Mediated Isothermal Amplification) nucleic acid amplification techniques.