Molecular Diagnostics of Porcine Stress Syndrome Susceptibility Associated with the Arg615Cys Mutation Using Real-Time PCR with Fluorescent Hybridization Probes

Abstract

Objective

The purpose of the present study was to develop a molecular genotyping method test by using a real time PCR hybridization probe and applying it to the analysis of C1843T mutations of the Sus scrofa RYR1 gene.

Animals population Three PSS-susceptible and PSS non-susceptible crossbred swine races were used for the experiments: Pietrain X Landrace Belga, Pietrain X Large White and Pietrain X Duroc.

Methods

We have developed a genotyping method by using a hybridization probe and applied it to the analysis of C1843T mutations of the RYR1 gene, associated with PSS susceptibility. Genotyping results obtained by hybridization probe strategies were confirmed by restriction analysis and sequencing. In addition, phenotype/genotype correlation analyses were developed by using the in vitro contracture test and confirmed the in vivo halothane-succinylcholine challenge.

Results

The real-time PCR with fluorescent hybridization probe methodology was designed to identify homozygous PSS-resistant, PSS-susceptible animals as well as heterozygous carriers. All cases genotyped by fluorescent hybridization probes were in agreement with PCR restriction enzyme digestion and sequencing and showed a 100% concordance between the in vivo and in vitro porcine stress syndrome (PSS) susceptibility results.

Conclusions and clinical relevance

The real-time PCR with fluorescent hybridization probe method described here provides a rapid, easily interpretable and reliable tool for genotyping the C1843T (Arg615Cys) polymorphism of the RYR1 gene. This new methodology may be useful in the wide-scale genotyping of PSS-susceptibility and genetic selection.