Development and validation of PCR-RFLP assays for genotyping PRNP and PRND polymorphisms in New Zealand rabbits in Vietnam

作者：Bui APN, Thong LQ, Duong CN, Van Quang T, Giao NDM · 发表于：Research in veterinary science · 年份：2026 · DOI：10.1016/j.rvsc.2026.106215 · 研究领域：Polymorphism, Restriction Fragment Length、Polymerase Chain Reaction、Prions、Polymorphism, Genetic、Animals、Rabbits、Vietnam、Genotype

Genetic variation within the prion gene cluster (PRNP-PRND) has been proposed as a contributing factor to the prion-disease biology of rabbits, motivating the development of scalable genotyping tools for population screening and downstream genotype-phenotype studies. Here, we developed and validated PCR-restriction fragment length polymorphism (PCR-RFLP) assays to genotype one PRNP variant (c.234C > T) and two PRND variants (c.525G > C and c.579 A > G) in New Zealand rabbits from Vietnam. Blood samples were collected from 40 rabbits originating from a single commercial farm maintained under uniform husbandry conditions. Genotypes inferred by PCR-RFLP were confirmed by Sanger sequencing, which yielded high-quality chromatograms and unambiguous discrimination of homozygous versus heterozygous states. Across the cohort, the reference homozygote predominated at all three loci, and alternative alleles were detected only in heterozygous form, specifically, PRNP c.234C > T, 36/40 CC and 4/40 CT (q(T) = 0.050); PRND c.525G > C, 37/40 GG and 3/40 GC (q(C) = 0.0375); and PRND c.579 A > G, 35/40 AA and 5/40 AG (q(G) = 0.0625). Hardy-Weinberg testing indicated no significant deviation from equilibrium for any locus (all p > 0.05). In addition, chromatogram review identified an extra variant, c.637G > A, observed as a heterozygous G/A call (IUPAC "R"), highlighting the utility of sequence confirmation for uncovering additional polymorphisms within the amplified regions. Collectively, thes...