Separation of endogenous viral elements from infectious Penaeus stylirostris densovirus using recombinase polymerase amplification

• PCR detection of Penaeus stylirostris densovirus (PstDV) is problematical.
• RPA-based assay was developed for primary detection of PstDV.
• RPA assay is twice as fast, specific and sensitive for the detection of PstDV.
• RPA assay is 10 fold more sensitive than a non-nested PCR.
• The method is suitable for screening in both laboratory and field application.

Non-infectious Penaeus stylirostris densovirus (PstDV)-related sequences in the shrimp genome cause false positive results with current PCR protocols. Here, we examined and mapped PstDV insertion profile in the genome of Australian Penaeus monodon. A DNA sequence which is likely to represent infectious PstDV was also identified and used as a target sequence for recombinase polymerase amplification (RPA)-based approach, developed for specifically detecting PstDV. The RPA protocol at 37 °C for 30 min showed no cross-reaction with other shrimp viruses, and was 10 times more sensitive than the 309F/R PCR protocol currently recommended by the World Organization for Animal Health (OIE) for PstDV diagnosis. These features, together with the simplicity of the protocol, requiring only a heating block for the reaction, offer opportunities for rapid and efficient detection of PstDV.