Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis

Sullivan, Benjamin P.; Chou, Yu-Shan; Bender, Andrew T.; Martin, Coleman D.; Kaputa, Zoe G.; March, Hugh; Song, Minyung; Posner, Jonathan D.

doi:10.1101/2022.01.11.475898

Cited by 2 publications

(4 citation statements)

References 50 publications

(58 reference statements)

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“…Indeed, the formation of RPA clusters that correlate with the starting DNA concentration has been reported recently. 30 To examine the formation of RPA clusters, we visualize the DNA products using an intercalating dye (EvaGreen) during RPA under high magnification (100× magnification). Upon amplification, we observe the emergence of irregular aggregates with a broad size distribution and spanning up to 10 μm in effective diameter (Figure 1A,B).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Valloly

Roy

2022

Anal. Chem.

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Amplification-based quantitative polymerase chain reaction (qPCR) provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to many settings. Quantitative isothermal amplification methods alleviate the need for thermal cyclers; however, they still require continuous monitoring of the nucleic acid amplification on sophisticated readers. Here, we adapted an isothermal recombinase polymerase amplification (RPA) reaction to develop a semiquantitative method that relies on the final amplicon yield to estimate the initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and showed that the amplicon yields at the end of the RPA reaction correlated well with the starting DNA concentration while reducing nonspecific amplification robustly. We demonstrate this approach, termed quantitative endpoint RPA (qeRPA), to detect DNA over five log orders with a detection limit of 100 molecules. Using a linear regression model of the normalized endpoint intensity (NEI) standard curve, we estimate the viral load from the serum of dengue virus-infected patients with comparable performance to qPCR. Unlike the conventional isothermal quantitative methods, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field deployment in resource-limited settings.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…We speculated that such aggregation would be driven by the local accumulation of new amplicons fueled by rapid DNA amplification during RPA. Indeed, the formation of RPA clusters that correlate with the starting DNA concentration has been reported recently …”

Section: Resultsmentioning

confidence: 99%

Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Valloly

Roy

2022

Anal. Chem.

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show abstract

“…Indeed, formation of RPA clusters that correlate with starting DNA concentration has been reported recently. 28 To examine the formation of RPA clusters, we visualize the DNA products using an intercalating dye (EvaGreen) during RPA under high magnification (100X magnification). Upon amplification, we observe emergence of irregular aggregates with a broad size distribution and spanning up to 10 µm in effective diameter (Figure 1A, B).…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, formation of RPA clusters that correlate with starting DNA concentration has been reported recently. 28…”

Section: Resultsmentioning

confidence: 99%

Nucleic acid quantification with amplicon yield in recombinase polymerase amplification

Valloly

Roy

2022

Preprint

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Amplification-based qPCR provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to different settings. Here, we adapted isothermal Recombinase Polymerase Amplification (RPA) reaction to develop a semi-quantitative method that relies on final amplicon yield to estimate initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and show that the amplicon yields at the end of the RPA reaction correlates well to the starting DNA concentration while reducing non specific amplification robustly. We demonstrate this approach termed here as quantitative endpoint RPA (qeRPA) to detect DNA over five log orders with detection limit of 100 molecules. Using a linear regression model with normalized endpoint intensity (NEI) standard curve, we estimate viral load from the serum of dengue-infected patients with comparable performance to qPCR. Hence, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field-deployment in limited-resource settings.

show abstract

Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis

Cited by 2 publications

References 50 publications

Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Nucleic acid quantification with amplicon yield in recombinase polymerase amplification

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