A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

Gavin J Nixon; Helle F Svenstrup; Carol E Donald; Caroline Carder; Judith M Stephenson; Stephen Morris-Jones; Jim F Huggett; Carole A Foy

doi:10.1016/j.bdq.2014.11.001

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

Biomol Detect Quantif. 2014 Nov 22:2:4-10. doi: 10.1016/j.bdq.2014.11.001. eCollection 2014 Dec.

Authors

Gavin J Nixon¹, Helle F Svenstrup², Carol E Donald¹, Caroline Carder³, Judith M Stephenson⁴, Stephen Morris-Jones⁵, Jim F Huggett¹, Carole A Foy¹

Affiliations

¹ LGC, Queens Road, Teddington, UK.
² Awapatent A/S, Copenhagen, Denmark.
³ Medicine Clinical Board, University College London Hospitals NHS Foundation Trust, London, UK.
⁴ Institute for Women's Health, University College London, London, UK.
⁵ Department of Clinical Microbiology, University College London Hospitals NHS Foundation Trust, London, UK.

Abstract

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Keywords: Cq, quantification cycle; Diagnostics; IDT, isothermal doubling time; Isothermal nucleic acid amplification; MIQE, minimum information for the publication of quantitative real-time PCR experiments; NAA, nucleic acid amplification; Quantitative LAMP; Quantitative real time PCR; Standardisation; Tt, threshold time; qLAMP, quantitative loop-mediated amplification; qPCR, quantitative real-time polymerase chain reaction; td, doubling time.