* freight costs are not included
a) A collection of 103 samples was used to validate primarily this kit gently provided by Dr. Parra F. (Universidad Oviedo). This collection included 69 liver samples which contained RHDVb confirmed by sequencing, 23 samples with RHDV (G1), 2 samples of RHDV (G6) and 9 negative samples. Concordance was almost perfect yielding a Kappa value of 0.978.
b) Ten RHDVb positive sample collected in different locations in Italy were also analyzed by the OIE Reference Laboratory for RHD at Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna. All ten samples resulted RHDV2 positives with the EXOone qPCR kits.
c) Twenty three RHDVb rabbit livers collected in different locations of UK were firstly confirmed as positive by conventional PCR and sequencing and then also resulted positive with EXOone qPCR kit ranging a Cq value from 6 to 19.
d) An exclusivity test was performed with 22 pathogens which affect rabbits including main bacteria (Chlamydophila abortus, Escherichia coli, Listeria monocitogenes, Pasteurella multocida, Salmonella, Staphylococcus aureus…), virus (Myxoma virus) and parasites like Encephalitozoon cuniculi. The classic RHDV (G1) isolate Ast/89 RNA was also tested. None of the 22 pathogens tested resulted positive.
e) 190 clinical cases were tested with this qPCR. Specimens such as liver (n=180) and feces (n=10) were analyzed individually or conforming pools up to 5 animals. Cq values ranging from 12.35 to 27.78.
• We studied the reportable range for this qPCR assay using a purified specific synthetic oligonucleotide (RABI positive control).
• This qPCR kit can identify RHDVb from clinical samples.
• Maximum quantification limit of RHDVb ≥1.2x1010 copies/rxn.
• Minimum quantification limit of RHDVb = 1.2x102 copies /rxn.