Double-Loop-Mediated Isothermal Amplification (D-LAMP) using colourimetric gold nanoparticle probe for rapid detection of infectious Penaeus stylirostris densovirus (PstDNV) with reduced false-positive results from endogenous viral elements

Endogenous viral elements (EVEs) of Penaeus stylirostris densovirus (PstDNV) in shrimp genome have been reported to occur randomly, and may cause false positive in diagnosis, because most of conventional detection methods only target a small nucleotide sequence of PstDNV. Since PstDNV is listed by the World Organization for Animal Health (OIE) as a reportable virus in crustacean, several countries require shrimp stocks imported for aquaculture to be screened for this virus. The rapidly and highly sensitive detection methods with reduced false positive from EVEs are necessary to guarantee and maintain absence of infectious PstDNV in traded stocks. To overcome problem posed by the viral insertions to shrimp genome, two set of primers were designed for LAMP combined with colourimetric gold nanoparticles (AuNPs), so-called D-LAMP, to detect two regions of PstDNV genome. The first target region targeted OIE-recommended nucleotide sequence of 1740-1968 (GenBank Accession no 273215) from our previous report. The second region targeted at the 3' end of the PstDNV genome that has been reported least likely to give rise to EVE in the shrimp genome. Detection sensitivity is approximately 100 copies. Results from LAMP combined with colourimetric AuNPs and conventional PCR methods are in agreement for detecting the infectious PstDNV. D-LAMP is also applicable to detect infectious type of PstDNV in P. stylirostris tissue sections (i.e. in situ DIG-labelled LAMP) and reaffirms its high specificity for detecting PstDNV infection.