Development of a Recombinase Polymerase Amplification (RPA) assay for acute hepatopancreatic necrosis disease (AHPND) detection in Pacific white shrimp (Penaeus vannamei)

Abstract

Acute hepatopancreatic necrosis disease (AHPND) is currently the most important bacterial disease of shrimp that has caused enormous losses to the shrimp industry worldwide. The causative agent of AHPND are Vibrio spp. Carrying plasmids containing the pirA and pirB genes which encode binary toxins, PirAB. Currently, AHPND is mostly diagnosed by PCR-based platforms which require the use of sophisticated laboratory instrumentation and are not suitable for a point-of-care diagnostics. Therefore, the availability of an alternative method based on isothermal amplification would be suitable for AHPND detection outside a laboratory setting and extremely useful at a pond side location. Isothermal amplification is based on the nucleic acid amplification at a single temperature and does not require the use of a thermal cycler. In this study, we developed an isothermal Recombinase Polymerase Amplification (RPA) assay for AHPND detection targeting both pirA and pirB genes, simultaneously and evaluated the specificity and sensitivity of the assay. The assay could detect AHPND without any cross-reaction with other microbial pathogens and Specific Pathogen Free (SPF) shrimp. The limit of detection of the assay was 5 copies of pirAB genes. To evaluate the reliability of the assay in detecting AHPND, DNA from Penaeus vannamei shrimp displaying acute and chronic infection were analyzed by the RPA assay and the results were compared with SYBR Green real-time PCR assay. While there was a 100% conformity between the two assay while detecting acute phase infection, RPA appeared to be more sensitive in detecting chronic phase infection. The data suggest that RPA assay described here would be a reliable method in detecting AHPND outside a standard laboratory setting.