A NEW NESTED PCR METHOD FOR EARLY DETECTION OF PATHOGENIC Vibrio spp. CAUSING ACUTE HEPATOPANCREATIC NECROSIS DISEASE IN SHRIMP

Acute hepatopancreatic necrosis disease (AHPND) is a disease  in shrimp caused by infection with toxic Vibrio strains, predominantly Vibrio parahaemolyticus, that harbor a plasmid containing the binary toxin genes pirA and pirB. This disease not only causes significant damage to the aquaculture industry but can also lead to antibiotic misuse, which poses environmental risks. Because the essential genes responsible for Vibrio‘s pathogenicity (pirA/B)  are located on the plasmid, detection of AHPND based solely on the presence of Vibrio parahaemolyticus alone is not accurate, as it does not assess whether the bacteria carry a plasmid containing the pirA/B genes. Currently, the PCR method using the AP3 primer, the standard method for detecting AHPND-causing Vibrio in Vietnam (TCVN 8710-19:2019), is a single-step PCR method with limited sensitivity. Thus, a new high-sensitivity nested PCR method was developed in this study for early AHPND detection by targeting the toxin genes. The AP5 primer set was specifically designed for amplification of the pirA-pirB region in the pVPA3-1 plasmid (GB: KM067908.1), with product size of 1,970 bp for the 1st round and 433 bp for the 2nd round of amplification, respectively. The adjusted concentration of outer primers was 0.3 pmol/µL. The optimized annealing temperatures for the outer primers and inner primers were 69 ºC and 70 ºC, respectively. Optimized reactions can detect as few as 103 plasmid copies per reaction. The sensitivity of this method was 100 times higher compared to PCR with AP3 primers and 1,000 times higher compared to AP4 method. Such a significant improvement in sensitivity opens up the possibility of developing these optimized reactions into a commercial kit that would enable the direct detection of AHPND using samples with low bacterial loads, such as larval shrimp and farming pond water.