Comparison of molecular detection PCR methods for chequa iflavirus in freshwater crayfish, Cherax quadricarinatus

Sakuna, Kitikarn, Elliman, Jennifer, and Owens, Leigh (2018) Comparison of molecular detection PCR methods for chequa iflavirus in freshwater crayfish, Cherax quadricarinatus. Journal of Virological Methods, 251. pp. 139-144.

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Chequa iflavirus (+ve sense ssRNA virus) infects redclaw crayfish (Cherax quadricarinatus) and it may cause mortality reaching 20-40% after about three weeks following stress. The sequence of the RNA-dependent RNA polymerase at nucleotide position 8383-9873 was used for developing and comparing PCR-based detection protocols. The reverse transcription, quantitative, polymerase chain reaction (RT-qPCR) was specific against nine Picornavirales and crustacean viruses and its' measurement of uncertainty (0.07-1.37) was similar to PCRs for other crustacean viruses. In vitro, the reverse transcription loop-mediated isothermal amplification (RT-LAMP) read at 60 min had poor repeatability for a linearized plasmid with an iflavirus insert when compared with RT-PCR visualised on an electrophoretic gel and RT-qPCR; both sensitive to 10(2) copies. In a limited, comparative sample of clinical crayfish haemolymph, the lowest, non-zero copies were 2.88 x 10(4) for RT-PCR and 4.60 x 10(1) for the RT-qPCR. In 68 further clinical crayfish haemolymph samples tested by RT-qPCR only, copy numbers ranged from 0 to 1.14 x 10(6). For RT-qPCR, the amplification plots, melt curves and the C-T values indicated that the C-T above 34.0 is a potential negative result but examination of the melt curve is necessary for an accurate interpretation. A suggested program of testing for crayfish farmers would consist of non-destructive bleeding, labelling of crayfish and screening with RT-qPCR. Only those crayfish nominally negative (below detectable limits) would be used for broodstock or selective breeding.

Item ID: 51816
Item Type: Article (Research - C1)
ISSN: 1879-0984
Keywords: chequa iflavirus, redclaw crayfish, Cherax quadricarinatus, stressed, RT-qPCR, RT-LAMP
Copyright Information: © 2017 Elsevier B.V. All rights reserved.
Funders: Australian Government: Rural Industries Research Development Corporation,, James Cook University JCU, North Queensland Crayfish Farmers
Projects and Grants: PRJ-009388
Date Deposited: 20 Dec 2017 07:31
FoR Codes: 31 BIOLOGICAL SCIENCES > 3107 Microbiology > 310706 Virology @ 90%
30 AGRICULTURAL, VETERINARY AND FOOD SCIENCES > 3005 Fisheries sciences > 300501 Aquaculture @ 10%
SEO Codes: 83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830101 Aquaculture Crustaceans (excl. Rock Lobster and Prawns) @ 80%
97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 20%
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