Development of a visual rapid detection method for aquatic animal-derived Trypanosoma via MIRA-LFD

Trypanosoma is a genus of flagellated protozoan parasites that commonly infect the blood of various vertebrates, including aquatic animals. Under natural conditions, these pathogens are vectored by leeches and may induce clinical manifestations such as anemia, edema, and appetite suppression in hosts. Prolonged infection can lead to severe weight loss, ultimately contributing to economic losses in aquaculture. In this study, we developed a rapid diagnostic assay combining Multienzyme isothermal rapid amplification (MIRA) with Lateral flow dipstick (LFD) visualization for detecting aquatic animal-derived Trypanosoma. Using Small Subunit Ribosomal DNA (SSU rDNA) gene as the detection target, specific primers and probes were designed. The optimal primer and probe combination were selected via primer screening. Through the optimization of reaction conditions and specificity and sensitivity testing, a method for detecting trypanosomes in aquatic animals was established using MIRA-LFD. The optimal reaction temperature for MIRA is 45℃. The reaction time is 15 min, and the result observation time is 5 min, resulting in a total duration of the entire detection process of 20 min. The specificity test results indicated that the method can specifically detect trypanosome DNA, with no cross-reactions observed with eight common parasites of aquatic animals (e.g., Argulus sp., Cryptocaryon irritans) or five host species (e.g., large yellow croaker, koi carp). The sensitivity test results demonstrated a detection limit of 8 pg/µL for trypanosome DNA (1000-fold higher than conventional PCR) and could detect as few as 1 trypanosome, compared to 100 trypanosomes required for conventional PCR. This method successfully detected trypanosome DNA from various tissue samples of large yellow croaker (Larimichthys crocea), including gill mucus, blood, and kidney tissue. In contrast, the PCR method only detected trypanosome DNA in blood samples. Clinical validation with 24 samples revealed 100 % detection rate for positive samples by MIRA-LFD, outperforming PCR's 83.33 % (10/12) rate. The MIRA-LFD method developed in this study for detecting aquatic animal-derived trypanosomes is rapid, simple, highly sensitive, and specific. It requires no sophisticated or expensive equipment, making it ideal for basic laboratories and on-site testing.