Colorimetric detection of scale drop disease virus in Asian sea bass using loop-mediated isothermal amplification with xylenol orange

Abstract

Scale drop disease virus (SDDV) is an emerging pathogen that causes multiple outbreaks in farmed Asian sea bass across many regions in Southeast Asia and results in severe economic losses to the exports of fish. With no current standard vaccination protocol against this virus, routine testing that probes for SDDV in Asian sea bass culture can help farmers prevent a large-scale epidemic from occurring. Fish samples can be sent to accredited laboratories for SDDV test that is performed via polymerase chain reaction (PCR) and related methods, such as qPCR. Albeit its high sensitivity, PCR requires skilled labors and equipment only available in centralized laboratory settings, and therefore, not appropriate for the timely determination of SDDV. Herein, we report the development of a colorimetric assay based on loop-mediated isothermal amplification (LAMP) with a pH-sensitive xylenol orange (XO) reporter. With a rapid total DNA extraction method developed in this research, we were able to demonstrate that the sensitivity of the naked-eye detection was 100 picograms of total DNA extracted from the SDDV-infected Asian sea bass samples. In addition to its affordability, our LAMP-XO circumvents the technical challenges faced by PCR and allows the SDDV detection to be conveniently performed and analyzed at the potential site of an outbreak.