Leptospirosis is a waterborne zoonotic disease caused by pathogenic species of the genus Leptospira that affects humans and animals. Current diagnostic methods, such as culture and serology-based techniques, have limitations, including low sensitivity and time-consuming nature. This study presents the first implementation of the polymerase spiral reaction (PSR) assay for the rapid and visual detection of Leptospira interrogans. The assay utilizes Lipl32, a highly conserved gene that encodes an outer membrane lipoprotein, as a marker gene to detect pathogenic Leptospira spp. For visual detection, the assay was evaluated using a colorimetric dye and nucleic acid stain. The assay was optimized for various reaction parameters. The optimal conditions were determined to be 60°C for 60 min with 6U of Bst Polymerase, 5 mM of Mg2+, 2.5 mM of Betaine, and 10 mM of dNTPs. An artificial contamination study in tap water established the assay’s detection limit at 16 GEq/mL. Notably, the PSR assay showed high specificity and sensitivity compared to loop-mediated isothermal amplification (LAMP), requiring only a single primer pair in contrast to the six primers required for LAMP.
Key words: Leptospira; Waterborne, Zoonosis, Polymerase Spiral Reaction, Lipl32
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