Development of Duplex Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue for Detection of Infectious Spleen and Kidney Necrosis Virus and Aeromonas hydrophila in Chinese Perch (Siniperca chuatsi)

Bacterial sepsis caused by <i>Aeromonas hydrophila</i> (<i>A. hydrophila</i>) and infectious spleen and kidney necrosis virus disease (ISKNVD) caused by infectious spleen and kidney necrosis virus (ISKNV) frequently result in significant mortality among Chinese perch (<i>Siniperca chuatsi</i>). Co-infection of mandarin fish with <i>A. hydrophila</i> and ISKNV occurs from time to time. In this study, a visual detection method for ISKNV and <i>A. hydrophila</i> was developed, using loop-mediated isothermal amplification (LAMP) and pre-addition of hydroxynaphthol blue. Primers for amplifying LAMP in the same system were designed based on the conserved regions of the MCP gene of infectious spleen and kidney necrosis virus, as well as the <i>hlyA</i> gene of <i>A. hydrophila</i>. The results showed that this method amplified bright trapezoidal bands in the presence of only <i>A. hydrophila</i> or ISKNV and both, with sky blue for positive amplification and violet for negative amplification. There was no cross-reactivity with other pathogens, and fragments of 182 bp, 171 bp and 163 bp appeared after digestion of the <i>A. hydrophila</i> LAMP product and 136 bp, 117 bp and 96 bp appeared after digestion of the ISKNV LAMP product. This holds true even when both positive products are present simultaneously. The minimum detection limit of this method was 100 fg for <i>A. hydrophila</i> and 100 fg for ISKNV, and the minimum detection limit for the mixed template was 1 pg. Overall, this method has high sensitivity and specificity to rapidly detect and distinguish between the two pathogens.