AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus

Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VPAHPND) revealed that it was mediated by a binary Pir-like toxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPND-causing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences of ToxA, a one-step PCR detection method for VPAHPND was introduced in June 2014 but had the limitation that attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VPAHPND in shrimp or other samples could not be detected without first preparing an enrichment broth culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VPAHPND using a panel of 104 bacterial isolates including 51 VPAHPND isolates and 53 non-AHPND isolates, the latter including 34 isolates of V. parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species. The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template) than the one-step AP3 (10 pg total DNA template) method, and it could detect VPAHPND in experimentally challenged shrimp by 6 h post immersion (n = 2/3), while AP3 could not detect is until 12 h post immersion (n = 1/3). Thus, the AP4 method may be useful in detecting VPAHPND isolates in samples where target material is limited (e.g., small tissue quantity or archived DNA) and enrichment cannot be employed (i.e., frozen samples or samples preserved in alcohol).