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

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.

Fig. 1

Target sequences with corresponding amplicon regions for qLAMP (shaded sequence) and qPCR assays (underlined sequence). (a) Chlamydia trachomatis plasmid DNA for growth within mammalian cells (GenBank Acc#X07547) 1081–1560 bp target region for qPCR and qLAMP assay. (b) M. genitalium partial MgPa gene (strain M2300) (GenBank Acc#X91072) 161–480 bp target region for qPCR and qLAMP assay.

Fig. 2

Standard curve based analysis of qPCR and qLAMP cryptic plasmid/MgPa assay performance. Plots displaying mean C_q [(a) & (b)] and T_t [(c) & (d)] from three separate reactions per three experiments when threshold set at the same respective fluorescence. Error bars illustrate standard deviations.

Fig. 3

Comparative analysis of qPCR and qLAMP assay amplification success. Plots displaying qPCR [(a) & (b)] and qLAMP [(c) & (d)] successful amplification data from three separate reactions per 3 experiments (n = 9).

Fig. 4

Methodologies to compare general assay performance. (a) Percentage PCR efficiency, (b) Isothermal Doubling Times. Plots displaying IDT or PCR efficiency (n = 3 separate experiments, 3 technical replicates). Error bars denote inter-experimental standard deviations.