A new highly sensitive lateral flow immunochromatographic assay for the detection of PirB toxin from acute hepatopancreatic necrosis disease-causing Vibrio species

Abstract

Acute hepatopancreatic necrosis disease (AHPND) is a severe condition that has caused significant damage and substantial financial losses to the global shrimp industry. Consequently, developing a convenient, sensitive, and specific analytical assay for accurately detecting AHPND is crucial for controlling the spread of the disease. The etiologic agent of AHPND is a strain of Vibrio species that carries a plasmid encoding the virulent binary toxins PirA and PirB. In this study, a pair of novel high-affinity monoclonal antibodies (MAbs) targeting the PirB toxin was generated and employed to develop a highly sensitive lateral flow immunochromatographic assay (LFIA). The LFIA results are visible to an unaided eye within 15 min of applying the bacterial colonies or pre-enrichment sample prepared in the application buffer to the test strip. This assay exhibited high specificity, successfully identifying AHPND-causing Vibrio species (Vibrio parahaemolyticus and Vibrio campbellii) without cross-reacting with non-AHPND Vibrio species or other bacterial isolates examined. The assay demonstrated a detection limit of approximately 40 ng/ml for PirB toxin, representing a 100-fold improvement over the sensitivity of the previous test strip for PirB toxin. Notably, the detection limit was further enhanced after a 6-h pre-enrichment process, enabling the identification of toxin-producing bacteria at concentrations as low as 1 colony-forming unit (CFU)/ml. Thus, it achieves a detection limit comparable to that of one-step PCR assays. Due to its high specificity, ease of use, rapid results, and no requirement for specialized equipment, the LFIA strip is an ideal tool for monitoring AHPND outbreaks and accurately detecting AHPND infections in shrimp cultures.