Rapid detection of decapod iridescent virus 1 by recombinase polymerase amplification combined with CRISPR-Cas12a

Abstract:

[Background] Decapod iridescent virus 1 (DIV1), which can infect Penaeus vannamei, Penaeus chinensis, Penaeus japonicus, etc., is one of the main viruses harmful to the prawn aquaculture. Currently, the efficient, rapid, and simple detection of whether the prawn is infected with DIV1 is an effective way to reduce the occurrence and damage of the virus. [Objective] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas12a), termed CRISPR-Cas12a system, was combined with recombinase polymerase amplification (RPA) to establish a method RPA-Cas12a for the rapid detection of DIV1, and then the application value of this method in detecting actual samples was assessed. [Methods] We extracted the DNA of DIV1, and designed the RPA primers of DIV1, crRNA, and reporting probes to establish the rapid detection method. Subsequently, we analyzed the sensitivity and specificity of this method in detecting DIV1 and compared the consistency of the established method with qPCR. [Results] RPA-Cas12a can detect the DIV1 DNA samples within 40 min and had the sensitivity of 10 copies/reaction. This method only detected DIV1 while showed negative results for white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, and Enterocytozoon hepatopenaei. Moreover, the RPA-Cas12a method revealed consistent positive rate with qPCR in detecting 61 actual samples. [Conclusion] The RPA-Cas12a method established in this study is rapid, simple, sensitive, and specific, and can serve as a new tool for the rapid detection of DIV1.

Key words:CRISPR-Cas12a;recombinase polymerase amplification;Decapod iridescent virus 1;rapid detection