A novel PCR method for simultaneously detecting Acute hepatopancreatic Necrosis Disease (AHPND) and mutant-AHPND in shrimp

Abstract

Acute Hepatopancreatic Necrosis Disease (AHPND) is a globally serious threat, especially for Vietnam shrimp culture. This disease is caused by toxin-carrying bacteria Vibrio parahaemolyticus which plasmid encodes binary toxins ToxA and ToxB. The disease has a mortality rate by up to 100% after 3–5 days post infection and fast spread among shrimp culturing regions. In 2017, a mutant of this disease named mutant-AHPND was recorded with a 1053 bp transposon inserted into toxA gene, toxB gene was not affected but the mortality rate was still up to 50% and the disease still spread quickly. There is no PCR test which can simultaneously detect both the normal and mutant plasmids of this disease commercially available. In this study, we developed a PCR test with single primer pairs which can specifically and simultaneously detect both types of the disease in shrimp. The PCR technique is established with a sensitivity of 2.5 pg per 20 μl reaction for plasmid solution and is four-times higher than that of the AP3 PCR kit which is commercially used in detection of AHPND disease in shrimp. Whereas the specificity of the method analyzed using two Vp-AHPND strains, one mutant Vp-AHPND strain, thirteen non-AHPND Vibrio species and White spot syndrome virus (WSSV) revealed that the method does not give unspecific reaction with non-AHPND bacteria and WSSV. The established PCR reaction is the core step in developing a PCR kit that helps detect both types of disease-causing plasmid in shrimp. With optimized parameters, this PCR kit can also be a cost-effective and rapid tool which can be used by shrimp cultivators and organizations in disease detection for epidemic monitoring.