Rapid and Quantitative Loop-mediated Isothermal Amplification (LAMP) Assays for Discriminatory Detection of Vibrio cholerae

Abstract

Cholera outbreaks continue to pose significant public health challenges, particularly in resource-constrained regions of Africa and Southeast Asia. Limitations in existing surveillance tools and approaches are one impediment to rapid and effective public health responses. To address this, we developed molecular beacon-based loop-mediated isothermal amplification (LAMP) assays targeting Vibrio cholerae (V. cholerae) markers ompW, O1rfb, O139rfb, and tcpA relevant to species-, serogroup-, and biotype-level detection. The assays achieved high analytical sensitivity with limits of detection ranging from ~99 to 487 gene copies per reaction, performed in duplex with minimal change in non-specific background, and were robust when tested on wastewater as a complex environmental matrix. Quantitatively, the assays showed a strong monotonic association between the logarithmic target concentration and the assay’s time-to-threshold (Tt) (Spearman’s ρ: consistently high across targets, ρ < -0.53), but only moderate linearity (single-plex R2 = 0.59 to 0.74, duplex R2 = 0.54 to 0.72). While the monotonic relationship is strong, concentration estimates remain uncertain, constraining precise linear quantification. We outline assay design and analysis features that help reconcile these differences and may guide future improvements. Despite some limitations, particularly variability in time-to-reach threshold at low target concentrations, these validated LAMP assays show potential as tools for cholera surveillance and outbreak response . However, further optimization, including improving reproducibility at low concentrations and minimizing false positives during extended reaction times, would enhance their reliability for routine field deployment.