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

Area of Science:

• Environmental DNA (eDNA) analysis
• Molecular biology techniques
• Aquatic ecology

Background:

• eDNA analysis demands sensitive and specific methods due to low target DNA concentrations.
• Conventional LAMP assays can produce nonspecific amplification products.
• TaqMan probes can improve LAMP specificity and sensitivity for ecological applications.

Purpose of the Study:

• To develop and evaluate a TaqMan probe-based LAMP assay for detecting Oryzias latipes eDNA.
• To assess the sensitivity, specificity, and compatibility with simple extraction methods of the TaqMan LAMP assay.

Main Methods:

• Development of a TaqMan probe-based loop-mediated isothermal amplification (LAMP) assay.
• Testing analytical sensitivity and specificity against nontarget species.
• Evaluation with crudely extracted eDNA using the suspended glass fiber method.

Main Results:

• The TaqMan LAMP assay demonstrated higher analytical sensitivity (approx. tenfold) compared to turbidimetric LAMP.
• The assay detected low copy numbers of template DNA and functioned with minimally processed eDNA.
• Specificity testing confirmed accurate detection of O. latipes without cross-reactivity with other fish species.

Conclusions:

• The TaqMan LAMP assay offers a sensitive and species-specific method for O. latipes detection in environmental samples.
• Its compatibility with simple DNA extraction supports practical, rapid biomonitoring.
• Further field studies are needed to confirm performance advantages over existing LAMP methods.