Development of a Visual Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Morganella morganii

Abstract

Morganella morganii is a Gram-negative bacterium associated with foodborne diseases and histamine toxicity in fish, posing significant public health concerns. Traditional detection methods, such as polymerase chain reaction (PCR), often require costly equipment, restricting their applicability in resource-limited environments. In this study, we present the development of a cost-effective and user-friendly colorimetric loop-mediated isothermal amplification (LAMP) assay. We designed six primers targeting the urease gene and optimized the assay to produce visual results using a pH-sensitive phenol red dye. The LAMP assay delivers results in 25 min and can detect M. morganii at concentrations as low as 5 pg (1.16 × 10⁶ copies/reaction) and 2.6 × 10⁵ CFU/mL (2.5 × 10⁶ copies/reaction) in pure cultures, demonstrating high specificity, with no cross-reactivity against 10 other bacterial strains. A comparison with PCR methods using 30 samples revealed a 100% concordance rate, indicating robust diagnostic agreement. Additionally, the assay successfully identified M. morganii in spiked aquaculture water samples at concentrations up to 10⁷ CFU/mL. In summary, this LAMP assay operates at a constant temperature and provides clear visual results, serving as a potential rapid detection method for applications in aquaculture settings.