Reproducibility crisis in isothermal amplification: lessons from benchmarking LAMP assays

Area of Science:

• Microbiology and Molecular Diagnostics
• Biotechnology and Assay Development

Background:

• Isothermal amplification assays, including loop-mediated isothermal amplification (LAMP), have proliferated for pathogen detection.
• Lack of standardized reporting guidelines hinders reproducibility and comparability of these assays.
• Previous publications often present incomplete assay composition and performance data.

Purpose of the Study:

• To systematically evaluate nine published loop-mediated isothermal amplification (LAMP) assays for Pseudomonas aeruginosa detection.
• To assess the adequacy of reported assay composition for implementation and reproducibility.
• To rigorously evaluate the performance (specificity, sensitivity, limit of detection) of these LAMP assays.

Main Methods:

• Benchmarking of nine published LAMP assays against qPCR.
• Screening for basic functionality, analytical specificity (using 19 non-target strains), sensitivity (using 13 P. aeruginosa strains), and limit of detection (LOD).
• Comparison of experimentally determined performance metrics with originally reported data.

Main Results:

• Four assays lacked essential composition details; most failed to report DNA concentrations or replicate numbers.
• Analytical specificity was overestimated in original publications; only 3 of 9 assays maintained >90% specificity in this study.
• Sensitivity was confirmed for two assays, but LOD values were significantly higher than reported, with qPCR outperforming all LAMP assays.

Conclusions:

• Significant discrepancies exist between published and experimentally validated performance of LAMP assays.
• Incomplete reporting and lack of standardized formats undermine the transparency, utility, and replicability of isothermal amplification assays.
• Enhanced publication standards and quality controls are crucial for reliable assay translation into clinical and environmental applications.