Truncation‐Enhanced Aptamer Binding Affinity and Its Potential as a Sensor for Macrobrachium rosenbergii Nodavirus Detection

ABSTRACT White tail disease in Macrobrachium rosenbergii is caused by M. rosenbergii nodavirus (MrNV) infection, resulting in up to 100% mortality in larvae and post‐larvae stages, severely impacting aquaculture production. Existing genome‐based detection methods for MrNV are costly and time‐consuming, highlighting the need for rapid and cost‐effective diagnostic tests. This study evaluated the effects of truncating selected aptamer on its binding affinity to the MrNV capsid protein. The previously isolated and identified aptamer through magnetic‐capture SELEX and Next Generation Sequencing demonstrated high binding affinity to the MrNV capsid protein. Truncation at the primer overhang was found to improve binding affinity, reducing the dissociation constant from 347 nM to 30.1 nM. The calculated limit of detection for the truncated aptamer decreased from 5.64 nM to 1.7 nM, while the limit of quantification decreased from 17.1 nM to 5.16 nM. These reductions indicate that the truncated aptamer has higher sensitivity compared to the full‐length aptamer. In tests with MrNV‐infected M. rosenbergii samples, both the enzyme‐linked aptamer assay and the gold nanoparticle aptasensor assay showed consistent results when 0.5 μg of total protein lysate was used. This indicates that the prawn protein concentration interferes with the detection of the viral protein. These findings suggest the potential application of the truncated aptamer as a sensor in the development of a practical aptamer‐based diagnostic kit. For instance, an aptamer‐based lateral flow assay test kit could provide a user‐friendly, cost‐effective solution that eliminates the need for sophisticated instrumentation for diagnosis or data interpretation, making it ideal for detecting MrNV infection in M. rosenbergii aquaculture.