Development of a real-time loop-mediated isothermal amplification (real-time LAMP) assay for the onsite detection of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) in shrimp

Acute hepatopancreatic necrosis disease (AHPN) is a lethal shrimp disease caused by Vibrio parahaemolyticus carrying virulent pir^AB genes in extrachromosomal plasmids. However, the limitations of current polymerase chain reaction (PCR) methods pose challenges for on-site AHPND diagnosis. This study aimed to develop a real-time Loop-Mediated Isothermal Amplification (LAMP) assay for diagnosing AHPND at shrimp farming sites. Two primer sets were designed to target a 300 bp region within the pir^AB gene on the pVPA3-1 plasmid of the reference strain 13–028/A3. Using a Genie II machine, an AHPND-specific primer set was selected to optimize a LAMP reaction mixture (LAMP-mixture I) and reaction conditions. The LAMP-mixture I provided clear and accurate results at 65 °C within approximately 50 min. The detection limit (100 fg) was comparable to that of duplex PCR developed for AHPND detection and was 100 times more sensitive than conventional PCR with existing LAMP primers (10 pg), though less sensitive than AP4 nested PCR. To enhance the feasibility of the LAMP assay in shrimp farms, a portable real-time LAMP machine developed by SMTION (Daejeon, Korea) was employed. The LAMP products were analyzed using SYBR Green I and calcein detection methods. Both methods produced positive results and showed no cross-reactivity with non-AHPND strains. The real-time LAMP calcein method demonstrated high diagnostic specificity, positive predictive value, and 78 % accuracy when evaluated with field samples. Hence, the real-time LAMP calcein method developed here offers potential for rapid and reliable AHPND diagnosis in shrimp farming sites, in comparison to other PCR-based strategies in simplicity and specificity.