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

• Published in: Journal of virological methods 2021-07, Vol.293, p.114169-114169, Article 114169
• Main Authors: Jaroenram, Wansadaj, Hayakijkosol, Orachun, Owens, Leigh, Elliman, Jennifer
• Format: Article
• Language: English
• Subjects: Australian Cherax quadricarinatus; Cherax reovirus; Real-time PCR; Redclaw crayfish
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2021.114169

•Gold standard method for Cherax reovirus detection is not previously available.•RT-qPCR is developed for the detection of Cherax reovirus.•The method is 100-times more sensitive than conventional PCR.•The method is 2-times faster than conventional PCR.•The method can serve as a gold standard for the detection of Cherax reovirus. 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.