Enhanced Specificity in Colorimetric LAMP Assay for Sarocladium kiliense Detection Using a Combination of Two Additives

Abstract

The genus Sarocladium comprises fungal species closely related to Acremonium, with S. kiliense and S. strictum being medically important. These species can cause infections in both immunocompetent and immunocompromised individuals. The current detection methods are limited, prompting the need for rapid and specific diagnostic tools. We developed a colorimetric loop-mediated isothermal amplification (LAMP) assay targeting S. kiliense (SK-LAMP). The initial prototype assay faced challenges with cross-reactivities with closely related species. To address this, we incorporated two additives, pullulan and tetramethylammonium chloride (TMAC), which are known to reduce non-specific signals in amplification assays. Our study found that a combination of 1% (v/v) pullulan and 0.03 M TMAC enhanced the specific detection of S. kiliense in a 45 min reaction, without non-specific false-positive results for other fungal species. This optimised LAMP assay demonstrated high sensitivity and specificity, offering a reliable and rapid method for detecting S. kiliense. The novel approach of combining additives to enhance assay specificity presents a promising strategy for improving LAMP assays targeting closely related fungal species. This advancement can aid in the timely diagnosis and management of infections caused by S. kiliense, contributing to better patient outcomes and infection control.