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Co-infection of mandarin fish with A. hydrophila and ISKNV occurs from time to time. In this study, a visual detection method for ISKNV and A. hydrophila 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 hlyA gene of A. hydrophila. The results showed that this method amplified bright trapezoidal bands in the presence of only A. hydrophila 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 A. hydrophila 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 A. hydrophila 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.<\/jats:p>","DOI":"10.3390\/microorganisms13030586","type":"journal-article","created":{"date-parts":[[2025,3,4]],"date-time":"2025-03-04T05:41:51Z","timestamp":1741066911000},"page":"586","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["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)"],"prefix":"10.3390","volume":"13","author":[{"given":"Xiao","family":"He","sequence":"first","affiliation":[{"name":"College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingyi","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xu","family":"Tan","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunan","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiguang","family":"Kong","sequence":"additional","affiliation":[{"name":"Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9796-8215","authenticated-orcid":false,"given":"Xiaodan","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China"},{"name":"International Research Laboratory of Prevention and Control of Important Animal Infectious Diseases and Zoonotic Diseases of Jiangsu Higher Education Institutions, Yangzhou University, Yangzhou 225012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1007\/s10228-018-0648-1","article-title":"Ontogenetic development and otolith microstructure in the larval and juvenile stages of mandarin fish Siniperca chuatsi","volume":"66","author":"ReferencesSong","year":"2019","journal-title":"Ichthyol. 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