Reproducibility crisis in isothermal amplification: lessons from benchmarking LAMP assays

The number of published isothermal amplification assays has increased substantially in recent years. Unfortunately, no harmonized guidelines, such as the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, exist for publishing these methods, often resulting in incomplete reporting of assay composition and performance. In this study, we systematically evaluated nine published loop-mediated isothermal amplification (LAMP) assays for the detection of Pseudomonas aeruginosa. We aimed to assess whether publications provide (i) sufficient information on assay composition to allow implementation and reproduction and (ii) robust data on assay performance to evaluate their applicability. Assays were screened for basic functionality, analytical specificity, sensitivity, and limit of detection (LOD) in head-to-head experiments with qPCR. Four assays lacked essential composition details, and almost all did not report DNA concentrations or replicate numbers. Only six assays consistently amplified target DNA. Analytical specificity testing with 19 non-target strains contradicted previously reported 100% specificity, with only 3 maintaining specificity above 90% in our evaluation. Sensitivity testing with 13 P. aeruginosa strains confirmed 100% sensitivity for two assays. However, LOD experiments revealed significantly higher values than originally reported, with qPCR outperforming all LAMP assays. These findings highlight substantial discrepancies between published data and real-world assay performance. The absence of standardized formats, consistent units, and complete methodological details undermines replicability. Although this study focused on P. aeruginosa, the identified issues are widely relevant across different microbial targets. We advocate for increased publication standards and quality controls to ensure transparency, utility, and comparability of isothermal amplification assays and to support their translation into clinical and environmental applications.IMPORTANCEOver the past decade, numerous isothermal amplification-based assays for the detection of pathogens or health-relevant microorganisms have been proposed, each claiming progress from the state-of-the-art and applicability to both clinical specimens and/or environmental samples. However, many published assays lack essential methodological details, and reported performance metrics are often inconsistent or incomparable. Using Pseudomonas aeruginosa as a representative model organism, we set out to test whether all details required for implementing assays were provided by original publications and if important assay characteristics were reproducible. The results of this systemic benchmarking of nine published loop-mediated isothermal amplification assays revealed major discrepancies between reported and experimentally measured performance in terms of specificity, sensitivity, and limit of detection. By highlighting the crucial elements that need to be reported, this work aims to improve the transparency, reproducibility, and overall quality of isothermal amplification assays, fostering their broader application across different research settings.