Abstract
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 is mainly caused by Vibrio species carrying the pVA1 plasmid encoding the virulence genes Photorhabdus insect-related (pir) pirVPA and pirVPB. We developed a new molecular assay that combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology (RPA-CRISPR/Cas12a) to detect pirVPA and pirVPB, with a fluorescent signal result. The fluorescence RPA-CRISPR/Cas12a assay had a detection limit of 20 copies/μL for pirVPA and pirVPB. To improve usability and visualize RPA-CRISPR/Cas12a assay results, a lateral flow strip readout was added. With the lateral flow strip, the RPA-CRISPR/Cas12a assay had a lower limit of detection of 200 copies/μL (0.3 fmol/L). The lateral flow assay can be completed in 2 h and showed no cross-reactivity with pathogens causing other shrimp diseases. In a field test of 60 shrimp samples, the RPA-CRISPR/Cas12a lateral flow assay showed 92.5% positive predictive agreement and 100% negative predictive agreement. 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.
Highlights
As the demand for shrimp continues to increase worldwide, Asia has become a dominant supplier of farmed penaeid shrimp, in particular Penaeus vannamei (P. vannamei) and Penaeus monodon (P. monodon)
As the first step in establishing the new CRISPR/Cas12a assay for Acute hepatopancreatic necrosis disease (AHPND) (Figure 1), Cas12a cleavage activity was assessed in an in vitro cleavage assay
The results of the in vitro cleavage assay are shown in Figure 1B, intact dsDNA template (1,794 bp) was cleaved by Cas12a in the presence of crRNA, indicated that all four crRNAs enable Cas12a to cleave the target DNA template, crRNA3 induced a specific ontarget cleavage with the expected cleave products of 1,142 and 652 bp, respectively, while the other crRNAs did not
Summary
As the demand for shrimp continues to increase worldwide, Asia has become a dominant supplier of farmed penaeid shrimp, in particular Penaeus vannamei (P. vannamei) and Penaeus monodon (P. monodon). Several groups have pursued molecular testing to detect AHPND by targeting pirVPA and pirVPB, using methods including polymerase chain reaction (PCR), nested PCR, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and real-time quantitative PCR (qPCR) [19,20,21,22]. We developed a novel molecular assay that combines the CRISPR/Cas12a system with RPA and lateral flow technology to detect AHPND virulence genes in cultured penaeid shrimp (P. vannamei and P. monodon). Our new molecular assay (LFS-based RPA-CRISPR/Cas12a) has the potential to detect AHPND with high accuracy without the need for lab equipment, providing a convenient and simple method that can be deployed to the field for rapid AHPND detection in cultured shrimp
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