Enterocytozoon hepatopenaei (EHP) is one of the most serious pathogens in shrimp farming. This study combines recombinase polymerase amplification (RPA) with the Argonaute from Pyrococcus furiosus (PfAgo) and establishes a sensitive and reliable method for on-site detection of EHP. With careful screening of gDNA and optimization of the reaction, the method shows a good specificity and reaches a sensitivity of single copy per reaction, which is higher than the sensitivity of the currently available molecular assays. The whole procedure can be finished within 1.5 h including the sample processing time and only requires minimum laboratory support, which is user-friendly for on-site environments. This is the first application of PfAgo for the diagnosis of infectious diseases in seafood supply chains. It provides a reliable method for on-site detection of EHP in shrimp farms and establishes a groundwork for multiplex detection of important pathogens in seafood farming using PfAgo.
Acute hepatopancreatic necrosis disease (AHPND) is one
of the most
devastating diseases in aquaculture, causing significant economic
losses in seafood supplies worldwide. Early detection is critical
for its prevention, which requires reliable and fast-responding diagnosis
tools with point-of-care testing (POCT) capacity. Recombinase polymerase
amplification (RPA) has been combined with CRISPR/Cas12a for AHPND
diagnosis with a two-step procedure, but the operation is inconvenient
and has the risk of carryover contamination. Here, we develop an RPA-CRISPR
one-pot assay that integrates RPA and CRISPR/Cas12a cleavage into
simultaneous reactions. Using the special design of crRNA, which is
based on suboptimal protospacer adjacent motifs (PAM), RPA and Cas12a
are made compatible in one pot. The assay is highly specific with
a good sensitivity of 102 copies/reaction. This study provides
a new choice for AHPND diagnosis with a POCT facility and sets a good
example for developing RPA-CRISPR one-pot molecular diagnosis assays.
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