Establishment of pre-enrichment step and DNA extraction method for the novel PCR procedure of acute hepatopancreatic necrosis disease (AHPND) and mutant-AHPND fast detection in shrimp

After the first outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) in China in 2009, this disease has been still considered a globally dangerous disease in shrimp aquaculture industry. Currently, there is no effective method to prevent and treat AHPND. Therefore, rapid detection methods which could avert and control this disease in shrimp were considered the most effective strategy. In 2021, a new PCR reaction was established that can simultaneously detect AHPND and mutant-AHPND. Intending to develop a PCR kit, the PCR procedure including a pre-enrichment step and DNA-extracted method was established to conduct the PCR reaction. The new PCR procedure was verified with a detection limit of 5.10^3 CFU/mL. This detection limit was two times higher than the conventional PCR methods, which are currently used to detect AHPND. Vibrio parahaemolyticus showed the best growth at 37°C in the broth with hepatopancreas of shrimp. A simple boiling method to extract DNA in bacterial broth with shrimp tissues was also modified. The PCR procedure has successfully been validated on 42 AHPND- suspecting samples. These results would be developed into a novel standard procedure using a PCR kit for rapid detection of AHPND and related mutant-AHPND for rapid disease diagnosis in shrimp farms.