American journal of veterinary research · 2007
Development of quantitative polymerase chain reaction assays for allelic discrimination of gangliosidoses in cats.
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Wang Chi-Young J, Smith Bruce F
Аннотация
OBJECTIVE: 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.
SAMPLE POPULATION: DNA samples obtained from archived feline blood samples submitted for GM1 and GM2 testing.
PROCEDURES: 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.
RESULTS: 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.
CONCLUSIONS AND CLINICAL RELEVANCE: 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.
SAMPLE POPULATION: DNA samples obtained from archived feline blood samples submitted for GM1 and GM2 testing.
PROCEDURES: 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.
RESULTS: 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.
CONCLUSIONS AND CLINICAL RELEVANCE: 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.