Developing a TaqMan loop-mediated isothermal amplification assay for Oryzias latipes detection

Environmental DNA (eDNA) analysis requires highly sensitive and specific detection methods because target DNA is often present at low concentrations among diverse biological materials. Although LAMP is a rapid and robust amplification technique, conventional assays can generate nonspecific products. Incorporating TaqMan probes into LAMP reactions may enhance both specificity and sensitivity for ecological monitoring.

We developed a TaqMan probe-based loop‑mediated isothermal amplification (TaqMan LAMP) assay to detect Oryzias latipes from eDNA. Under controlled laboratory conditions, the assay showed approximately tenfold higher analytical sensitivity than turbidimetric LAMP and detected template DNA at low copy numbers in triplicate tests; however, this lowest detected concentration represents a preliminary estimate rather than a statistically defined limit of detection. Using crudely extracted eDNA obtained via the suspended glass fiber method, both TaqMan LAMP and turbidimetric LAMP detected O. latipes. These results indicate that TaqMan LAMP can function with minimally processed eDNA, although the present field data do not demonstrate improved inhibitor tolerance or superior performance relative to turbidimetric LAMP. Specificity testing confirmed that fluorescence signals were observed only for O. latipes and not for the tested nontarget fish species.

The TaqMan LAMP assay provides a sensitive and species‑specific approach for detecting O. latipes from environmental samples. Its compatibility with simple DNA extraction workflows highlights its potential for practical and rapid biomonitoring applications, although further field‑based evaluations will be required to clarify whether it offers performance advantages over existing LAMP methods.