Affiliation:
1. School of Breeding and Multiplication, Sanya Institute of Breeding and Multiplication, Hainan University, Sanya 572025, China
2. Collaborative Innovation Center of Marine Science and Technology, Hainan University, Haikou 570228, China
3. Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, School of Marine Biology and Fisheries, Hainan University, Haikou 570228, China
Abstract
Groupers are valuable economic fish in the southern sea area of China, but the threat of disease is becoming more and more serious. Vibrio harveyi, V. parahaemolyticus, and Singapore grouper iridovirus (SGIV) are three important pathogens that cause disease in groupers, and infection with either a single one or a mix of these pathogens poses a serious threat to the healthy development of grouper culture. To enhance the rapid diagnosis and screening in the early stages, it is necessary to develop rapid detection methods of these pathogens. To simultaneously and rapidly detect the three pathogens, in this study, we utilized the TolC of V. harveyi, DNAJ of V. parahaemolyticus, and RAD2 of SGIV as the target genes and established a triple visual loop-mediated isothermal amplification (LAMP) method. This LAMP method showed a detection time as fast as 30 min and a high sensitivity of 100 fg/μL. Moreover, this method exhibited strong specificity and no cross-reaction with seven types of Vibrio and Staphylococcus aureus, as well as five common viruses in aquatic animals. Then, the LAMP products were enzymically cut, and three characteristic strips were used to identify the pathogen species. The results of the clinical trials demonstrated that the method could accurately and specifically detect V. harveyi, V. parahaemolyticus, and SGIV in grouper tissues. In summary, this study successfully established a triple visual LAMP rapid detection method for V. harveyi, V. parahaemolyticus, and SGIV. The method offers several advantages including simple equipment, easy operation, rapid reaction, high specificity, high sensitivity, and visual results. It is suitable for the early and rapid diagnosis of groupers infected with V. harveyi, V. parahaemolyticus, and SGIV, thereby providing useful technical support for further application in the large-scale disease surveillance of aquaculture animals.
Funder
Hainan Province Science and Technology Special Fund
Hainan University Collaborative Innovation Center Fund
earmarked fund for HNARS-Grouper
Reference36 articles.
1. The People’s Republic of China Ministry of Agriculture (2023). Fisheries Bureau. China Fishery Statistical Yearbook 2023, China Agriculture Press.
2. Progress in disease research of grouper aquaculture in China;Luo;Fish. Sci.,2013
3. Mechanism of oligochitosan improving non-specific immunity of Epinephelus fuscoguttatus (♀) × E. lanceolatu (♂);Liu;J. Fish. China,2022
4. Vibrio parahaemolyticus flagellin induces cytokines expression via toll-like receptor 5 pathway in orange-spotted grouper, Epinephelus coioides;He;Fish Shellfish Immunol.,2019
5. Isolation, identification and pathogenicity of Vibrio harveyi, the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus;Shen;J. Fish Dis.,2017