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

Jaroenram, Wansadaj, Hayakijkosol, Orachun, Owens, Leigh, and Elliman, Jennifer (2021) Establishing a gold standard method for the detection of Cherax reovirus using reverse transcriptase, quantitative, polymerase chain reaction. Journal of Virological Methods, 293. 114169.

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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.

Item ID: 67908
Item Type: Article (Research - C1)
ISSN: 1879-0984
Keywords: Real-time PCR, Cherax reovirus, Redclaw crayfish, Australian Cherax quadricarinatus
Copyright Information: Published Version: Copyright © 2021 Elsevier B.V. All rights reserved. Accepted Version: Open access in an Institutional Repository under a CC BY-NC-ND license after a 12 month embargo.
Funders: Rural Industries Research and Development Corporation (RIRDC)
Projects and Grants: RIRDC PRJ-009388
Date Deposited: 19 Jul 2021 02:05
FoR Codes: 31 BIOLOGICAL SCIENCES > 3107 Microbiology > 310706 Virology @ 50%
31 BIOLOGICAL SCIENCES > 3107 Microbiology > 310704 Microbial genetics @ 50%
SEO Codes: 28 EXPANDING KNOWLEDGE > 2801 Expanding knowledge > 280102 Expanding knowledge in the biological sciences @ 50%
10 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 1002 Fisheries - aquaculture > 100201 Aquaculture crustaceans (excl. rock lobster and prawns) @ 50%
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