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Full Dynamic Range Quantification using Loop-mediated Amplification (LAMP) by Combining Analysis of Amplification

Patrick Hardinge¹, James A H Murray²

¹Cardiff School of Biosciences, Biomedical Science Building, Museum Avenue, Cardiff, CF10 3AX, UK. hardingep@cardiff.ac.uk.

Scientific Reports

|January 24, 2020

Summary

This study introduces new quantification metrics for loop-mediated amplification (LAMP), enabling accurate measurement of nucleic acid targets across a wide range of concentrations without dilution. These novel methods improve molecular diagnostics by addressing limitations of existing techniques like qPCR and digital PCR.

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Area of Science:

Molecular Biology
Biotechnology
Nucleic Acid Quantification

Background:

Quantitative PCR (qPCR) and digital PCR (dPCR) face limitations in accurately quantifying nucleic acid targets at very low or high concentrations, often requiring sample dilution.
Existing methods struggle with a broad dynamic range, impacting the precision of molecular diagnostic assays.

Purpose of the Study:

To develop novel quantification metrics for loop-mediated amplification (LAMP) that bridge the gap between high and low template concentrations.
To establish accurate quantification methods for low copy numbers (1-10 copies) using LAMP, suitable for undiluted samples.

Main Methods:

Real-time monitoring of LAMP reactions using bioluminescent reporting (BART) and fluorescent dye binding.
Analysis of reaction timing variations at low copy numbers as a quantification indicator.
Utilizing artificial templates to confirm the fundamental nature of LAMP initiation variation.

Main Results:

Increasing variation in LAMP reaction timings at low copy numbers correlates with decreasing template concentration.
Quantification of targets between 1 and 10 copies was achieved with high accuracy, primarily using timing variance.
The observed timing variation is an intrinsic feature of the LAMP initiation mechanism, not dependent on displacement primers.

Conclusions:

Developed LAMP quantification metrics offer a solution for accurate molecular target measurement across a wide dynamic range.
These methods facilitate precise quantification in closed-tube, undiluted samples, enhancing molecular diagnostic capabilities.
LAMP-based ultra-quantification strategies hold significant potential for a comprehensive diagnostic approach.