AUTHOR=Li Chenglong , Lin Nan , Feng Zhihua , Lin Minhua , Guan Biyun , Chen Kunsen , Liang Wangwang , Wang Qiaohuang , Li Miaomiao , You Yu , Chen Qi 

TITLE=CRISPR/Cas12a Based Rapid Molecular Detection of Acute Hepatopancreatic Necrosis Disease in Shrimp

JOURNAL=Frontiers in Veterinary Science

VOLUME=8

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2021.819681

DOI=10.3389/fvets.2021.819681

ISSN=2297-1769

ABSTRACT=<p>Acute hepatopancreatic necrosis disease (AHPND), formerly called early mortality syndrome (EMS), causes high mortality in cultured penaeid shrimp, particularly <italic>Penaeus vannamei</italic> and <italic>Penaeus monodon</italic>. AHPND is mainly caused by <italic>Vibrio</italic> species carrying the pVA1 plasmid encoding the virulence genes Photorhabdus insect-related (<italic>pir</italic>) <italic>pir</italic><sup><italic>VP</italic></sup><italic>A</italic> and <italic>pir</italic><sup><italic>VP</italic></sup><italic>B</italic>. We developed a new molecular assay that combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology (RPA-CRISPR/Cas12a) to detect <italic>pir</italic><sup><italic>VP</italic></sup><italic>A</italic> and <italic>pir</italic><sup><italic>VP</italic></sup><italic>B</italic>, with a fluorescent signal result. The fluorescence RPA-CRISPR/Cas12a assay had a detection limit of 20 copies/μL for <italic>pir</italic><sup><italic>VP</italic></sup><italic>A</italic> and <italic>pir</italic><sup><italic>VP</italic></sup><italic>B</italic>. 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.</p>