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

Objective : The purpose of the presera study was to devélop a molecular genotyping method test by using a real time PCR hybridization probé and applying it to the analysis of C1843T mutations of the Sus scrofa RYR1 gene. Animáis 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 devéloped a genotyping method by using a hybridization probé and applied it to the analysis of C1843T mutations of the RYR1 gene, associated with PSS susceptibility. Genotyping results obtained by hybridization probé strategies were confirmed by restriction analysis and sequencing. In addi-tion, phenotype/genotype correlation analyses were devéloped by using the in vitro contracture test and confirmed the in vivo hálothane-succinylcholine challenge. Results: The real-time PCR with fluorescent hybridization probé methodology was designed to identify ho-mozygous PSS-resistant, PSS-susceptible animáis as well as heterozygous carriers. All cases genotyped by fluorescent hybridization probes were in agreement with PCR restriction enzyme digestión 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 probé method described here provides a rapid, easily interpretable and réliáble tool for genotyping the C1843T (Arg615-Cys) polymorphism of the RYR1 gene. This new methodology may be useful in the wide-scale genotyping of PSS-susceptibility and genetic selection.

Clasificación por métodos moleculares de cepas de Trypanosoma spp aisladas en Colombia y evaluación morfométrica / Classification by means of molecular methods of Trypanosoma spp stumps isolated in Colombia and morphometric evaluation

Mediante el empleo de técnicas de PCR-RFLP, se caracterizaron tres cepas de tripanosomas aisladas de bovinos de diferentes regiones de Colombia, a partir de amplificaciones de la región 18S de la subunidad ribosomal del rDNA del parásito, para lo cual se emplearon para el PCR inicial, los primers específicos: nF2 y nR3 y sobre el producto obtenido de 756 pb, se realizó un PCR semianidado con los primers NF2 y NR2 de la misma región, obteniéndose un producto 650 pb sobre el cual se realizó el análisis de restricción con las enzimas MspI y Eco57I,lo que permitió clasificar las tres cepas analizadas como Trypanosoma vivax, Tripanosoma evansi y Trypanosoma theileri. El estudio morfométrico permitió corroborar diferencias morfológicas entre los parásitos clasificados. Se discute el uso de la técnica de PCR-RFLPs como herramienta diagnóstica de la tripanosomosis bovina en Colombia...