Development of a thermostabilised triplex LAMP assay with dry-reagent four target lateral flow dipstick for detection of Entamoeba histolytica and non-pathogenic Entamoeba spp.

- The developed triplex LAMP-NALFIA assay is ready-to-use and cold-chain-free.
- The triplex LAMP reagent mix is thermostabilized and lyophilised.
- The LAMP assay is designed to simultaneously amplified three specific DNA targets.
- The biosensor is developed to facilitate interpretation of post-LAMP analysis.
- The triplex LAMP-NALFIA assay could detect as low as 10 Entamoeba trophozoites.

This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5′ end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.

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