Search

KR-102961190-B1 - Simultaneous Diagnostic Method and Kit for White Spot Disease and Acute Hepatopancreatic Necrosis Disease in Shrimp Using Rapid Immunochromatography

KR102961190B1KR 102961190 B1KR102961190 B1KR 102961190B1KR-102961190-B1

Abstract

The present invention relates to a technology capable of simultaneously diagnosing white spot disease (WSD) and acute hepatopancreatic necrosis disease (AHPND), which cause mass mortality in shrimp farms worldwide. More specifically, the invention relates to a diagnostic kit and method for on-site testing that can simultaneously detect white spot syndrome virus (WSSV) and PirA within 10 minutes when a kit is prepared and applied using 2H9 (gold condensation antibody), a monoclonal antibody specific to Photorhadus insect-related (Pir) toxin A, 2B4 (antibody used on test line 1), a monoclonal antibody specific to WSSV, and 2A4 (antibody used on test line 2), based on an immunochromatography method.

Inventors

  • 임창용
  • 최준범
  • 한승훈
  • 최보현
  • 김충남
  • 문인식
  • 신기욱
  • 김진숙

Assignees

  • 전라남도

Dates

Publication Date
20260508
Application Date
20251103
Priority Date
20250929

Claims (5)

  1. A monoclonal antibody characterized by being produced by hybridoma cell line 2A4 deposited under accession number KCLRF-BP-00574, hybridoma cell line 2B4 deposited under accession number KCLRF-BP-00575, or hybridoma cell line 2H9 deposited under accession number KCLRF-BP-00576, and showing positivity in a lateral flow immunochromatographic assay (LFIA).
  2. In claim 1, the monoclonal antibody produced by the hybridoma cell line 2H9 deposited under accession number KCLRF-BP-00576 is positive for both the antigen protein of White spot syndrome virus (WSSV) and Photorhadus insect-related (Pir) toxin A, and The monoclonal antibody produced by the hybridoma cell line 2A4 deposited under accession number KCLRF-BP-00574 is specific to Photorhadus insect-related (Pir) toxin A, and The monoclonal antibody produced by the hybridoma cell line 2B4 deposited under accession number KCLRF-BP-00575 is a monoclonal antibody characterized by being specific to the antigen protein of White Spot Syndrome Virus (WSSV).
  3. delete
  4. A strip-shaped nitrocellulose membrane; a sample pad located at the leftmost part of the nitrocellulose membrane; An antibody-conjugated gold pad formed laminated immediately to the right of the absorption pad of the above-mentioned nitrocellulose membrane and containing the monoclonal antibody 2H9 produced by hybridoma cells deposited under accession number KCLRF-BP-00576; Test line 1, on which monoclonal antibody 2B4 produced by hybridoma cells deposited under accession number KCLRF-BP-00575 is immobilized in the middle portion of the above nitrocellulose membrane, test line 2, on which monoclonal antibody 2A4 produced by hybridoma cells deposited under accession number KCLRF-BP-00574 is immobilized, and control line, on which an antibody binding to said monoclonal antibody is immobilized; An LFIA-based rapid diagnostic kit for point-of-care testing for the simultaneous detection of acute hepatopancreatic necrosis disease (AHPND) and White spot syndrome virus (WSSV) in shrimp, characterized by including an absorbent pad located at the far right portion of the nitrocellulose membrane.
  5. Hybridoma cell line 2A4 deposited under accession number KCLRF-BP-00574, or hybridoma cell line 2B4 deposited under accession number KCLRF-BP-00575, or hybridoma cell line 2H9 deposited under accession number KCLRF-BP-00576

Description

Simultaneous Diagnostic Method and Kit for White Spot Disease and Acute Hepatopancreatic Necrosis Disease in Shrimp Using Rapid Immunochromatography The present invention relates to a technology capable of simultaneously diagnosing white spot disease (WSD) and acute hepatopancreatic necrosis disease (AHPND), which cause mass mortality in shrimp farms worldwide. More specifically, the invention relates to a diagnostic kit and method for on-site testing that can simultaneously detect white spot syndrome virus (WSSV) and PirA within 10 minutes when a kit is prepared and applied using 2H9 (gold condensation antibody), a monoclonal antibody specific to Photorhadus insect-related (Pir) toxin A, 2B4 (antibody used on test line 1), a monoclonal antibody specific to WSSV, and 2A4 (antibody used on test line 2), based on an immunochromatography method. White spot syndrome (WSD) was first reported in Taiwan in the 1990s and has since been reported as a disease affecting crustaceans, particularly farmed shrimp, in Japan, China, Southeast Asia, South Asia, the Middle East, and the United States. In Korea, it was first isolated from farmed Japanese prawns ( Marsuoenaeus japonicus ) in 1993 and has since occurred in Japanese prawns ( Fenneropenaeus chinensis ) and whiteleg shrimp ( Litopenaeus vannamei ), causing serious damage. White spot syndrome virus (WSSV) is a rod-shaped virus with an outer membrane belonging to the genus Whispovirus in the family Nimaviridae , and consists of six major proteins (outer membrane proteins: VP28, VP19, nucleocapsid: VP664, VP26, VP24, VP15) and about 300 kbp (dsDNA) of nucleic acid. Acute hepatopancreatic necrosis disease (AHPND) is reported to be a bacterial disease that causes fatal damage to farmed shrimp. Since being reported in Asia in 2010, it has been reported in the United States in 2013 and in Africa in 2017. In some cases, AHPND results in a 100% mortality rate within 30 days of stocking the larvae. Main symptoms include abnormal swimming or swimming near the bottom of the pond, slowed growth, and a shrinking size of the hepatopancreas as it turns white. The exoskeleton also softens, and black spots or streaks appear on the hepatopancreas. Starting with China in 2009, the disease has been reported in Vietnam, Malaysia, and Thailand; with the Philippines recently classified as a benign country, disease management is urgently needed in Korea as well. This disease is caused by infection with Vibrio parahaemolyticus, one of the bacteria that inhabit marine water. Vibrio parahaemolyticus is a representative marine bacterium that is isolated from estuaries and marine environments around the world, and it also exists in normal shrimp. Some possess heat-resistant enterotoxins and are therefore also known as 'Vibrio parahaemolyticus,' a pathogen that causes food poisoning in humans. Vibrio parahaemolyticus possesses a bacterial plasmid, within which exists a gene specifically designed to produce insect toxins. Since shrimp and insects are distant relatives, the modified insect toxins attack the hepatopancreas in shrimp, causing mortality. The infection route is primarily through the shrimp's oral cavity, where the toxins attach to and multiply within the stomach to form toxins. The toxins then travel from the stomach to the hepatopancreas, a major organ of the EMS, damaging the organs and preventing the shrimp from consuming or digesting food, ultimately leading to death. In the AHPND strain, the heat-resistant hemolysin-related gene that exhibits pathogenicity to humans was not expressed, but two genes involved in the Type VI secretion system (T6SS) (Pir A/B toxin genes) were expressed, and it was found that these genes are associated with the pathogenicity of AHPND. In addition, it is defined as "infection caused by Vibrio harveyi (V. harveyi) possessing a plasmid that produces Pirtoxin (PirA, PirB), as well as V. parahaemolyticus (VPAHPND), which was previously reported as the causative agent." In Korea, Vibrio parahaemolyticus ( VP AHPND ), which carries the Photorhadus insect-related (Pir) toxin, was detected for the first time in farmed whiteleg shrimp ( Litopenaeus vannamei ) in 2016. This disease mainly occurs during the early stages of farming (within 30 days of larvae), and the mortality rate can reach 70–100% within a few days. AHPND is primarily caused by specific transformed Vibrio parahaemolyticus . This strain possesses a plasmid approximately 70 kb in size, which contains the PirA and PirB genes (Photorhabdus insect-related toxins) and induces toxicity. These two toxins are similar to insecticidal toxins and destroy the hepatopancreatic cells of shrimp. Conventional methods for testing WSSV and VPAHPND include molecular biological methods [polymerase chain reaction (PCR), real-time PCR, etc.], immunological methods using antibodies [enzyme-linked immunosorbent assay (ELISA), fluorescent antibody techniques (IFAT, etc.], and histopathological methods. Looking at the adv