Establishment of Basic-RPA and RPA-LFD Rapid Detection Methods for Ranavirus in Largemouth Bass (Microterus...

Area of Science:

• Aquatic Virology
• Molecular Diagnostics
• Fish Disease Research

Background:

• Largemouth bass ranavirus (LMBRaV) causes significant mortality in largemouth bass aquaculture.
• Rapid and sensitive detection methods are urgently needed to prevent LMBRaV transmission and outbreaks.

Purpose of the Study:

• To develop and optimize basic recombinase polymerase amplification (basic-RPA) and RPA combined with lateral flow dipstick (RPA-LFD) assays for LMBRaV detection.
• To evaluate the sensitivity, specificity, and speed of the developed RPA assays.

Main Methods:

• Designed primers and probes targeting the mcp gene of LMBRaV.
• Optimized reaction conditions (temperature, time, primer concentration) for basic-RPA and RPA-LFD.
• Assessed specificity against other aquatic viruses and determined the detection limit.
• Compared RPA assay performance with the PCR assay using clinical samples.

Main Results:

• Established basic-RPA (25 min at 38°C) and RPA-LFD (15 min at 45°C) assays for LMBRaV.
• Both assays demonstrated high specificity, detecting only LMBRaV and not other tested aquatic viruses.
• Achieved a sensitive detection limit of 1 copy/μL DNA for both basic-RPA and RPA-LFD.
• RPA assays identified nine additional positive clinical samples compared to PCR.

Conclusions:

• Basic-RPA and RPA-LFD assays offer sensitive, rapid, and simple methods for LMBRaV detection.
• These assays are suitable for on-site diagnosis, aiding in the management of LMBRaV disease in aquaculture.
• The developed RPA methods improve upon traditional PCR in terms of speed and clinical sample detection.