Bates, A.; Li, J.; Rivero, F.; Wollenberg Valero, K. C.

Quantitative Loop-mediated isothermal amplification (qLAMP) is a relatively new method that has gained popularity in recent years, particularly in disease identification, including during the recent SARS-CoV-2 pandemic. Unlike conventional quantitative PCR (qPCR), qLAMP features a linear amplification phase before the exponential phase. Determining cycle threshold (Ct) values through automatic thresholding may therefore produce inaccurate results, and the nature of these thresholds complicates comparability between studies and softwares. We introduce a new method for transforming sigmoidal amplification curves into inflection threshold curves (iCt) to address issues with auto thresholds and analysis of qLAMP. This method is implemented as a collection of R functions named LAMPrey, suitable for analysis of both qPCR and qLAMP reactions performed in the two most commonly used real-time thermocyclers. We simulate qLAMP amplification differences, demonstrate that iCt and Ct methods perform equivalently for conventional qPCR with an Illumina library quantitation kit, and show that iCt values outperform Ct values for quantifying qLAMP reactions in zebrafish embryos. All scripts developed for this paper are available at https://github.com/dodged13/LAMPrey