Development of quantitative polymerase chain reaction assays for allelic discrimination of gangliosidoses in cats

Abstract

To develop quantitative PCR (qPCR) assays with allele-specific primers to provide a rapid and accurate diagnostic and screening test for the 3 mutations identified as causes of gangliosidoses in domestic cats. DNA samples obtained from archived feline blood samples submitted for GM1 and GM2 testing. A qPCR assay was developed for each mutation to monitor the efficiency of PCR amplification. Results were determined on the basis of the fluorescent intensity of DNA staining. Samples from 60 cats were screened by use of the 3 qPCR assays. Of these, 59 qPCR results agreed with the sequence-derived genotypes. The phenotype (affected) for the other cat agreed with results for the qPCR assay, which indicated that interpretation of the sequence-based result was incorrect. The qPCR assays offer a sensitive, rapid, and reproducible technique for allelic discrimination without the need for complicated processing steps, such as hybridization or sequencing, after PCR procedures. These assays may prove beneficial for a rapid diagnosis of gangliosidoses in cats and could also provide a means for reliable large-scale screening for the carrier state, thereby accelerating the eradication of these debilitating diseases from feline populations.