CRISPR/Cas12a-based Rapid Molecular Detection For Acute Hepatopancreatic Necrosis Disease (AHPND) In Shrimp

Acute hepatopancreatic necrosis disease（AHPND）,formerly called early mortality syndrome（EMS）,causes high mortality in cultured penaeid shrimp,particularly Penaeus vannamei and Penaeus monodon.AHPND infected shrimps show nearly 100%mortility and will deposit at the bottom of the pond.Due to the rapid spread of AHPND,the outbreak of the AHPND causes economic losses to the aquaculture industry worldwide.The pathogen of AHPND was identified as Vibrio species which including Vibrio parahaemolyticus,Vibrio owensii,Vibrio campbellii and Vibrio harveyi.AHPND infected shrimps are mainly resulted from Vibrio species carrying the p VA1 plasmid that encoding the virulence genes Photorhabdus insect-related（pir）pir^VPA and pir^VPB.When infected,the shrimps with AHPND present a shrinking hepatopancreas,and empty midgut and stomach.The high mortality of AHPND infected shrimp leads to the precipitous drop of the aquaculture production and business.To this end,in order to prevent the outbreak of the AHPND,timely assay of the AHPND infected shrimp is an effective method.Currently,the molecular diagnosis method of AHPND infected srhimp mainly targets virulence genes pir^VPA and pir^VPB.In this study,to distinguish from infected shrimp,we developed a new molecular assay that combines recombinase polymerase amplification（RPA）and CRISPR/Cas12a technology（RPA-CRISPR/Cas12a）to detect pir^VPA and pir^VPB through fluorescence assay and lateral flow strip assay,respectively.Firstly,the fluorescence RPA-CRISPR/Cas12a assay had a detection limit of 20 copies/μL for pir^VPA and pir^VPB.To improve usability and visualize RPA-CRISPR/Cas12a assay results,a lateral flow strip readout was added.Combined with lateral flow strip,the RPA-CRISPR/Cas12a assay had a lower limit of detection of 200 copies/μL（0.3 fmol/L）;After establishing the method,we then tested it with different pathogens from shrimp diseases,the results showed no cross-reactivity in these pathogens;In a field test of 60 shrimp samples,the RPA-CRISPR/Cas12a lateral flow assay showed 92.5%positive predictive agreement（PPA）and 100%negative predictive agreement（NPA）.As the new RPA-CRISPR/Cas12a assay is rapid,specific,and does not require complicated experimental equipment,it may have important field applications for detecting AHPND in farmed shrimp.