Development and Validation of a Multienzyme Isothermal Rapid Amplification Combined with Lateral-Flow Dipstick (MIRA-LFD) Assay for Trypanosoma Strains Circulating in Large Yellow Croaker (Larimichthys crocea)

Abstract

Trypanosomiasis, caused by flagellated protozoa of the genus Trypanosoma, has recently emerged as a major threat to aquaculture in China, particularly in farmed large yellow croaker (Larimichthys crocea). Outbreaks lead to high mortality rates and severe economic losses. Conventional diagnostic tools, such as blood-smear microscopy and molecular assays including polymerase chain reaction or quantitative polymerase chain reaction (qPCR), are often limited by low sensitivity during early infection or by their dependence on sophisticated instruments and trained personnel, restricting their utility in field conditions. To address these challenges, a multienzyme isothermal rapid amplification (MIRA) assay coupled with a lateral-flow dipstick (LFD) was developed for the rapid detection of trypanosoma strains circulating in L. crocea targeting the 18S ribosomal ribonucleic acid gene. After optimizing primer-probe sets, the assay performance was evaluated using plasmid standards and a panel of common aquaculture pathogens. The MRA-LFD assay consistently detected plasmid DNA at concentrations as low as 0.01 fg/µL (≈2.1 copies/µL) and demonstrated no cross-reactivity with other pathogens. Using clinical DNA samples positive for Trypanosoma, the detection limit was 100 fg µL−1. Validation with 150 tissue samples from fish with and without clinical symptoms demonstrated high diagnostic consistency (94%) with qPCR results, confirming the reliability of the assay. This MIRA-LFD platform provides a sensitive, specific and portable diagnostic tool for early detection of Trypanosoma infections in large yellow croaker, offering valuable support for surveillance and disease management in aquaculture.

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