Establishing a gold standard method for the detection of Cherax reovirus using reverse transcriptase, quantitative, polymerase chain reaction

Abstract

Cherax reovirus infects redclaw crayfish (Cherax quadricarinatus) and it may be involved in mortalities between 5-20 % and stunting of up to 40 % of survivors. The sequence of the RNA-dependent RNA polymerase was used to develop a reverse transcription, quantitative, PCR (RT-qPCR) which was specific against seven other crustacean viruses (Athtab bunyavirus, Chequa iflavirus, Macrobrachium rosenbergii nodavirus, Gill-associated virus, Taura syndrome virus, White spot syndrome virus, and Penaeus stylirostris Penstylhamaparvovirus) although GAV produced a reaction that was easily separated by melt curve analysis. A strong linear correlation (r² = 0.9965) was obtained between viral quantities ranging from 10⁷ to 10 viral copies/reaction with an amplification efficiency of 0.92. This RT-qPCR is 2-times faster and 100 times more sensitive than a standard RT-PCR using agarose gel electrophoresis with the potential to detect the virus down to 7.64 copies/reaction in clinical samples. In clinical crayfish samples, it was able to detect Cherax reovirus in crayfish when the traditional RT-PCR was negative. Its' measurement of uncertainty was less than 2% (0.02-1.9), similar to PCRs for other crustacean viruses. This RT-qPCR is proposed as the gold standard and should be used for the screening of populations of C. quadricarinatus for broodstock before being used in hatcheries or on farms.

Keywords: Australian Cherax quadricarinatus; Cherax reovirus; Real-time PCR; Redclaw crayfish.