On-site Detection of AHPND in Shrimp Farming by Probe-based Recombinase
Polymerase Amplification and Lateral Flow Strip
Abstract
Acute hepatopancreatic necrosis disease (AHPND) is an important
bacterial disease occurring early after stocking shrimp fry in shrimp
ponds with the mortalities of 100 %. AHPND leads to significantly drop
in production and brings out huge economic losses worldwide. Thus,
rapid, accurate, and convenient on-site detection method is urgent need
to monitor the outbreak and spreading of AHPND especially for
equipment-poor areas. Application of traditional PCR-based methods is
restricted due to the dependence on laboratory equipment and
technicians. In this study, an improved isothermal recombinase
polymerase amplification (RPA) combined lateral flow strip (LFS) assay
was developed for AHPND detection by introducing a probe. The specific
primers and probe were designed based on the PirAB gene, chemical
modifications were labelled to improve the specificity, and mismatched
bases were made to eliminate primer-dependent artifacts. In combination
with crude DNA extraction by boiling for 10 min, the RPA-LFS assay could
be finished within 25 min at 37-45°C and results were readable by naked
eyes. The exclusivity was validated to be no cross-reactivity with 10
other common vibrio spp strains. The inclusivity was verified using 10
other VPAHPND strains isolated from infected shrimps. The limit of
detection was 101 colony forming unit (CFU)/mL or 102 copies/μL and 100
CFU/10 g after 2 hours enrichment in spiked shrimp samples. The
detection accuracy was evaluated in a total of 75 collected shrimp and
seawater samples, which was proven to be consistent with AP4. The
established RPA-LFS method provides a rapid, accurate, sensitive and
equipment-free approach for on-site detection of AHPND and technical
references for monitoring other pathogens in cultivation industry.