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Establishing a gold standard method for the detection of Cherax reovirus using reverse transcriptase, quantitative, polymerase chain reaction
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Document Title
Establishing a gold standard method for the detection of Cherax reovirus using reverse transcriptase, quantitative, polymerase chain reaction
Author
Jaroenram W, Hayakijkosol O, Owens L, Elliman J
Name from Authors Collection
Affiliations
James Cook University; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
JOURNAL OF VIROLOGICAL METHODS
ISSN
0166-0934
Year
2021
Volume
293
Open Access
Green Accepted
Publisher
ELSEVIER
DOI
10.1016/j.jviromet.2021.114169
Format
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.
Funding Sponsor
Rural Industries Research Development Corporation, Australia [PRJ-009388]; North Queensland Crayfish Farmers association
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WOS