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.

Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP

“…As a consequence, a kinetic-based evaluation of an isothermal reaction's performance (e.g. an evaluation based on the proposed isothermal doubling time (IDT) parameter ( 42 )), would not discriminate between a slow, sensitive reaction, and a less sensitive (e.g. inhibited) reaction.…”

“…Reaction speed would seem to be an attractive criterion of efficiency when estimating isothermal amplification reaction performance ( 42 ) as well as when testing primers ( 43 ) and conditions ( 44 ) during optimization, as it is more convenient than performing dilutions and determining the LOD, the template concentration that can be detected with reasonable certainty, e.g. 95% confidence ( 5 ) for each condition.…”

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

“…Restriction enzyme gel electrophoresis [ 34 ], lateral flow biosensors [ 35 ] and pyrosequencing methods [ 36 ] have been used to demonstrate multiplex LAMP technology. These approaches however require laborious, contamination-prone, post-amplification analysis, preventing real-time quantifiable detection [ 37 ]. Multiplex LAMP based on antibody-antigen interactions has also been developed using immuno-chromatographic strips [ 38 ].…”

Advances in Directly Amplifying Nucleic Acids from Complex Samples

“…Isothermal amplification techniques like LAMP and RPA generally demonstrate a higher tolerance than PCR for urine, as they can be carried out directly. As such, LAMP-based assays without any pre-processing steps or chemical enhancements have detected the causative agents of viral infections [82,127,131] or STIs [222,223], and pathogenic bacteria such as Escherichia coli [224]. The developers of the recently established isothermal method SIBA showed the utility of this amplification technique by detecting Chlamydia trachomatis and Neisseria gonorrhoeae in a low-copy urine sample [25].…”