LAMPrey: a standardised method for analysing quantitative LAMP reactions using the inflection cycle threshold

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Quantitative loop-mediated isothermal amplification (qLAMP) is a growing gene expression quantification technique, vital for disease identification, as seen during the SARS-CoV-2 pandemic.
• qLAMP reaction kinetics can deviate from standard sigmoidal models, and the absence of ROX dye in some kits complicates analysis.
• Conventional cycle threshold (Ct or Cq) determination using auto-thresholding can lead to inaccuracies and hinder inter-study comparability.

Purpose of the Study:

• To introduce a novel method for transforming sigmoidal amplification curves into inflection cycle threshold curves (iCt).
• To address the limitations of auto-thresholding and improve the analysis of qLAMP data.
• To provide a robust tool for analyzing both qLAMP and quantitative PCR (qPCR) reactions.

Main Methods:

• Development of a new method to generate inflection cycle threshold (iCt) values from amplification curves.
• Implementation of the iCt method as a set of R functions named LAMPrey.
• Validation using simulated qLAMP data, conventional qPCR with an Illumina library quantitation kit, and a large-scale qLAMP experiment on zebrafish embryos.

Main Results:

• The iCt method demonstrated equivalent performance to the Ct method for conventional qPCR.
• iCt values significantly outperformed Ct values and the FDM metric in quantifying 2416 qLAMP reactions in zebrafish embryos.
• The LAMPrey R package provides a versatile tool for analyzing qLAMP and qPCR data from common real-time thermocyclers.

Conclusions:

• The iCt method offers a more accurate and reliable approach for determining gene expression levels from qLAMP data compared to traditional Ct values.
• LAMPrey facilitates improved analysis and comparability of qLAMP and qPCR results across different studies and software.
• This new method enhances the utility of qLAMP for applications requiring precise gene expression quantification.