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Real-time detection of the mutation responsible for progressive rod–cone degeneration in Labrador Retriever dogs using locked nucleic acid TaqMan probes

Correspondence: ¹Corresponding Author: Fabio Gentilini, Veterinary Clinical Department, Alma Mater Studiorum University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell'Emilia, Bologna, Italy. fabio.gentilini{at}unibo.it

Progressive rod–cone degeneration (prcd) is a late onset, autosomal recessive, inherited disease in dogs caused by a G > A substitution in the PRCD locus. prcd has been reported in more than 18 breeds, including Labrador Retriever dogs. In this study, a real-time polymerase chain reaction (PCR) assay, exploiting the features of locked nucleic acid (LNA) fluorescent-labeled probes, was developed to genotype the sequence variants responsible for the disease. Two Labrador Retrievers were diagnosed with prcd by ophthalmological examination performed by a panelist of the Italian hereditary eye disease control program. The 2 dogs, as well as 8 related and 14 unrelated Labrador Retrievers, were genotyped with both direct sequencing of the disease locus and real-time LNA TaqMan PCR assay. Even though the region surrounding the mutation was predicted to be highly structured, making probe annealing difficult, the real-time PCR assay allowed researchers to correctly genotype the dogs in all cases with a sensitivity threshold of 4 ng/reaction of genomic DNA. A real-time PCR assay will allow a high-throughput analysis of a larger cohort of dogs, thereby enabling researchers to investigate the prevalence of the mutated allele in the affected breeds.

Key Words: Dogs • locked nucleic acid • mutation • progressive rod–cone degeneration • real-time polymerase chain reaction