Development of Reverse Transcriptase Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick for Rapid Detection of Tilapia Lake Virus (TiLV): Pilot Study.

Abstract

Tilapia Lake Virus (TiLV) is well known as a highly contagious viral infection in aquaculture, particularly affecting Tilapia worldwide. Until recently, various TiLV diagnostic methods have been used for rapid and accurate diagnostic procedures that are crucial for timely disease detection and reducing losses. In this study, we developed an alternative method for investigating TiLV diagnosis using Reverse Transcriptase Recombinase Polymerase Amplification (RT-RPA) assay combined with a lateral flow dipstick (LFD). The test was generated by specific anti-FITC and anti-Biotin capture antibodies that are compatible with the TiLV-specific primers tagged with FITC and Biotin. The test was conducted by the reverse transcriptase of target TiLV RNA and RPA amplification at 39 °C for 20 min. The products were then determined by a positive band signal via LFD. The RT-RPA-LFD assay detected the plasmid of TiLV (pTiLV) with a Limit of Detection (LOD) of 3.19 copies/µL, while the RT-PCR-LFD assay detected it with an LOD of 319 copies/µL. Our findings demonstrate that RT-RPA-LFD represents a possible alternative to RT-PCR for the rapid and sensitive detection of TiLV, especially in areas with limited infrastructure.