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

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 60min 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 10² copies. In a limited, comparative sample of clinical crayfish haemolymph, the lowest, non-zero copies were 2.88×10⁴ for RT-PCR and 4.60×10¹ for the RT-qPCR. In 68 further clinical crayfish haemolymph samples tested by RT-qPCR only, copy numbers ranged from 0 to 1.14×10⁶. 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.