Establishment of triple-RPA-LFS detection method for three common shrimp viruses

Abstract

This study focuses on three viruses affecting farmed shrimp, including White Spot Syndrome Virus (WSSV), Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), and Taura Syndrome Virus (TSV). Specific primers and probes were designed by their respective conserved gene fragments to establish a triple-RPA-LFS detection method that simultaneously detects WSSV, IHHNV, and TSV. Seven pathogens and healthy shrimp tissues were collected to conduct specificity tests. This method can specifically amplify the gene fragments of WSSV, IHHNV, and TSV, while no fragments were amplified from the muscle tissues of healthy shrimp or other pathogens, indicating strong specificity. The reaction system was optimized, and specificity and sensitivity were validated. Sensitivity tests were conducted using a continuous dilution plasmid method, determining that the detection sensitivity of this method is 10¹ copies/reaction. Compared with the sensitivity of the qPCR detection method recommended by the World Organization for Animal Health (WOAH, formerly OIE), the triple-RPA-LFS method established in this study is faster and simpler to operate. When applied to test 110 samples simultaneously with the laboratory standard testing method, the results of the qPCR detection matched the results of the laboratory standard method with a 100 % concordance rate. These experimental results indicate that the triple-RPA-LFS detection method established in this study has the characteristics of high specificity, high sensitivity, short detection time, and high accuracy. It can be used for rapid on-site detection and diagnosis of the three pathogens: WSSV, IHHNV, and TSV.