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)

Zuo, You; Liao, Bichai; Lin, Luoxuan; Wu, Tong; Yuan, Jiahao; Xue, Qianxi; Wei, Haiyun; Liu, Shuming; Huang, Guangliang; Chen, Xinhua; Qin, Pan

doi:10.3390/fishes11020107

Open AccessArticle

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)

by

You Zuo

^1,†,

Bichai Liao

^2,†,

Luoxuan Lin

¹,

Tong Wu

¹,

Jiahao Yuan

¹,

Qianxi Xue

¹,

Haiyun Wei

¹,

Shuming Liu

¹,

Guangliang Huang

³,

Xinhua Chen

^1,4,* and

Pan Qin

^1,*

¹

State Key Laboratory of Mariculture Breeding, Key Laboratory of Marine Biotechnology of Fujian Province, College of Marine Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

²

Fishery Technology Promotion Station of Fujian Province, Fujian Agriculture and Forestry University, Fuzhou 350002, China

³

Fishery Technology Promotion Station, Fishery Department of Ningde, Ningde 352100, China

⁴

Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519000, China

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Fishes 2026, 11(2), 107; https://doi.org/10.3390/fishes11020107

Submission received: 3 December 2025 / Revised: 2 February 2026 / Accepted: 6 February 2026 / Published: 10 February 2026

(This article belongs to the Special Issue Microbial Pathogens, Disease Control and Veterinary Drug Use in Aquaculture)

Download

Browse Figures

Versions Notes

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.

Keywords: large yellow croaker; Trypanosoma sp.; multienzyme isothermal rapid amplification; lateral-flow dipstick

Share and Cite

MDPI and ACS Style

Zuo, Y.; Liao, B.; Lin, L.; Wu, T.; Yuan, J.; Xue, Q.; Wei, H.; Liu, S.; Huang, G.; Chen, X.; et al. 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). Fishes 2026, 11, 107. https://doi.org/10.3390/fishes11020107

AMA Style

Zuo Y, Liao B, Lin L, Wu T, Yuan J, Xue Q, Wei H, Liu S, Huang G, Chen X, et al. 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). Fishes. 2026; 11(2):107. https://doi.org/10.3390/fishes11020107

Chicago/Turabian Style

Zuo, You, Bichai Liao, Luoxuan Lin, Tong Wu, Jiahao Yuan, Qianxi Xue, Haiyun Wei, Shuming Liu, Guangliang Huang, Xinhua Chen, and et al. 2026. "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)" Fishes 11, no. 2: 107. https://doi.org/10.3390/fishes11020107

APA Style

Zuo, Y., Liao, B., Lin, L., Wu, T., Yuan, J., Xue, Q., Wei, H., Liu, S., Huang, G., Chen, X., & Qin, P. (2026). 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). Fishes, 11(2), 107. https://doi.org/10.3390/fishes11020107

Article Menu

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

Share and Cite

Article Metrics

Article Access Statistics

Further Information

Guidelines

MDPI Initiatives

Follow MDPI