Reverse transcription transcription polymerase chain reaction (RT-PCR) detection for Australian Cherax reovirus from redclaw crayfish (Cherax quadricarinatus)

Reoviruses have been isolated from many aquatic animals including fish and crustaceans. Viral inclusion bodies of reovirus have been found in the cytoplasm of the hepatopancreatic cells of redclaw crayfish (Cherax quadricarinatus). In the past, reverse transcription polymerase chain reaction (RT-PCR) designed from across other hosts was attempted to detect reovirus in redclaw crayfish but no specific set of primers successfully identified infected crayfish. In this study, two new sets of primers (Reo35F - Reo585R and Reo35F - Seq.R1) producing a 551 bp product and a 1370 bp product respectively were designed using C. quadricarinatus reovirus partial sequence (NCBI GenBank accession no. KM405245). After the removal of primer sequences, the smaller PCR product was a 99.21% match to KM405245 while the larger product was a 99.32% match. Only three amino acid differences were observed between the Australian and Chinese sequences. The Australian sequence is the ancestral sequence so changes are reported in that order: i.e. Australian>Chinese: 19Arg>Lys; 363Leu>Met; 423Gly>Asp. The longer Australian Cherax reovirus sequence inclusive of primer sequence has been submitted to NCBI GenBank number MN308286. The second set of primers was used in the world's first RT-PCR diagnostic method to detect the Australian reovirus from redclaw crayfish. The method has the detection limit of 1000 reovirus genome equivalents, and showed no cross-reactions with other prawn pathogens indicating its high sensitivity and specificity for Cherax reovirus diagnosis.