Noroviruses Surrogate Detection Using Loop-Mediated Isothermal Amplification (LAMP), Conventional RT-PCR and Quantitative RT-PCR in Sg. Tekala and Sg. Gabai Stream Water

Abstract

A preliminary study was carried out in order to evaluate a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detecting Noroviruses (NoV) RNA surrogates as external standard for NoV.The detection limit of the NoV RT-LAMP assay was observed to be 22 copies/μL. This RT-LAMP assay sensitivity was comparablewith the quantitative reverse transcriptase-Polymerase Chain Reaction (PCR) and shown to be 10-fold more sensitive than end-point conventional reverse transcriptase-PCR (RT-PCR). The NoV RT-LAMP assay showed high specificity to NoV targeted gene when specificity test was completed with no cross-reactivity with other 17 environmental strains. The assay also was performed with 11 spiked recreational stream water randomly picked from two recreational areas in Hulu Langat Malaysia, Sg. Tekala and Sg. Gabai. The RT-LAMP assay is simpler compared to the conventional PCR and real-time PCR, which in optimum isothermal temperature of 63°C, the amplification can be completed in 40 minutes. Results of spiked recreational stream water samples suggested that the NoV RT-LAMP assay can be used as monitoring tool for NoV surveillance in recreational stream water.