Mohamad Radzi, Nurhaslina and Mustaffa, Khairul Mohd Fadzli and Othman, Noratikah and Md. Isa, Muhammad Lokman and Jainlabdin, Mohd Hanif
(2025)
MNAZYME pan-fungal assay: an isothermal platform for broad-range fungal detection.
In: 1st International Biomedical Conference 2025, 3 - 4 September 2025, AC Marriot Hotel, Kuantan.
Abstract
INTRODUCTION: Fungal infections affect more than one billion individuals worldwide and are responsible for an estimated 1.5 million deaths each year. Delays in diagnosis, particularly in healthcare settings with limited resources, contribute to the severity of these outcomes. This study describes the development of a molecular assay using Multi-component Nucleic Acid Enzymes (MNAzymes) designed to detect conserved fungal ribosomal DNA sequences under isothermal conditions. OBJECTIVE(S): This study aimed to develop and evaluate a catalytic MNAzyme assay for universal detection of fungal DNA targeting conserved ribosomal regions. MATERIALS & METHODS: The assay targeted a conserved fungal sequence (AF-ITS4LR1) identified through multiple alignments of ITS and LSU regions. Custom Partzymes and a fluorogenic substrate (Sub3-JB) were designed to assemble on this target. Optimisation was performed for temperature (30–64 °C), Partzyme concentration (0.005–0.2 μM), and magnesium chloride (6–50 mM). Specificity was assessed using bacterial and non-target fungal controls. The limit of detection was determined via serial dilutions of the synthetic target sequence and fluorescence monitoring. RESULTS: The assay performed best at 50 °C, with 0.05 μM of each Partzyme, and 10 mM magnesium chloride. No fluorescence was detected in negative controls, indicating high specificity. The lowest detectable concentration of the synthetic target was 0.0025 μM, equivalent to approximately 3.76 x 1010 copies of the nucleic acid sequence. CONCLUSION: The developed MNAzyme assay enables specific, sensitive detection of fungal DNA under isothermal conditions and shows strong potential as a rapid, amplification-free diagnostic tool for fungal infections.
Actions (login required)
 |
View Item |
