Detection of rabbit hemorrhagic disease virus (RHDV), genotype GI.1 and GI.2 with a new multiplex real-time RT-qPCR protocol, using the minor capsid VP10 gene

Aim. This study aimed to develop a specific and sensitive multiplex real-time reverse transcription PCR (RT-qPCR) method for the detection and differentiation of rabbit hemorrhagic disease virus (RHDV) genotypes GI.1 and GI.2 circulating in Ukraine, using primers and probes based on the ORF2 gene, encoding the minor capsid protein VP10. Furthermore, to perform an initial validation of this PCR. Methods. The assay was designed to amplify a conserved 101-bp ORF2 sequence, encoding the minor capsid protein VP10, assuming that it would be a less variable region compared to that of the traditionally targeted ORF1 sequence encoding the major capsid protein VP60. Sequence alignment was performed using 38 full-genome sequences of RHDV isolates of various geographic origins present in GenBank. Specific primers to RHDV and two genotype-specific hydrolysis probes (FAM for genotype GI.1 and HEX for genotype GI.2) were designed and partially validated both in silico (BLAST) and in vitro. RNA was extracted from 6 tissue samples (contaminated with RHDV and negative control), and two virus reference strains using the IndiSpin Pathogen Kit, followed by RT-qPCR using a one-step protocol. Analytical sensitivity and specificity were assessed by a 10-fold RNA serial dilution (from 10⁶ to 10⁰ copies). For specificity testing, non-target organisms were included: Myxoma virus, Staphylococcus aureus, Pasteurella multocida, Escherichia coli, and Streptococcus agalactiae. Results. After optimization, the developed RT-qPCR assay demonstrated high analytical sensitivity, detecting as few as 100 RNA copies/reaction with consistent amplification across three replicates. The limit of detection (LOD) was established at 1.0×10² RNA copies for both genotypes. Standard curves yielded slopes of -3.44 (GI.1) and -3.38 (GI.2), corresponding to amplification efficiencies of 95.4% and 97.5%, respectively. The assay showed excellent linearity (R² = 0.925 for GI.1, R² = 0.881 for GI.2) and intra-assay variability (%CV) below 3.5%. No cross-reactivity was observed with non-target pathogens. Conclusions. The developed multiplex RT-qPCR assay demonstrated promising analytical performance for detecting and differentiating RHDV genotypes GI.1 and GI.2. These results provide partial validation of the method. However, limitations include testing only two genotypes, no internal amplification control, and lack of inter-laboratory comparison with WOAH-recommended VP60-based RT-PCR. Future studies should expand validation scope, incorporate internal controls, and perform multi-center validation before routine implementation.