Romanian Biotechnological Letters Vol.19, No2, 2014 Copyright © 2014 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER 9170 Romanian Biotechnological Letters, Vol. 19, No. 2, 2014 Screening of RYR1 genotypes in swine population by a rapid and sensitive method Received for publication, July 15, 2013 Accepted, February 26, 2014 DANIELA ELENA ILIE 1,2 , VASILE BĂCILĂ 3 , ADA CEAN 1,2 , LUDOVIC TOMA CZISZTER 2 , SIMINA NEO 2 1 Research and Development Station for Bovine - Arad, Arad, Romania 2 Banat’s University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies, Timisoara, Romania 3 University of Agronomic Science and Veterinary Medicine – Bucureşti, Faculty of Animal Science, Bucharest, Romania * Corresponding author: Vasile Băcilă, Phone: +40213192932, Fax: +40213512000 E-mail: vbacila@yahoo.com Abstract Genetic polymorphism in the gene controlling the calcium release channel (ryanodine receptor, RYR1) of sarcoplasmic reticulum in skeletal muscle have been shown to affect meat quality in pigs, producing porcine stress syndrome (PSS) and malignant hyperthermia (MH). The MH condition produces pale, soft and exudative (PSE) meat resulting in reduced commercial value of pork. Therefore, monitoring swine population for RYR1 genetic variants is important in the subsequent process of breeding and improving meat quality in pigs. The aim of the present study was to establish and validate a rapid and sensitive molecular genotyping assay to detect C1843T mutations of the Sus scrofa RYR1 gene using quantitative PCR (qPCR) followed by high-resolution melting (HRM) analysis. The qPCR-HRM assay allowed the rapid and sensitive identification of RYR1 genotypes for discrimination of wild type and mutant alleles, so the carriers can be detected. The method was validated by analyzing a total of 195 DNA samples that were previously genotyped by PCR-RFLP method. The results demonstrate that qPCR-HRM method is a fast, simple and reliable assay for genotyping the C1843T (Arg615Cys) polymorphism of the RYR1 gene. The high frequency of RYR1 mutant allele suggested that implementing a routine testing system is necessary to gradually eradicate the MH condition. Key words: C1843T SNP, high - resolution melting, malignant hyperthermia, molecular diagnostic, porcine stress syndrome, ryanodine receptor Introduction Ryanodine receptor (RYR1) is the calcium release channel from the sarcoplasmic reticulum in skeletal muscle whose contraction, relaxation and energy metabolism are regulated by the concentration of intracellular Ca 2+ (D.H. MACLENNAN & al. [1]). An abnormality in the ryanodine receptor facilitates opening and inhibits closing of Ca 2+ channel resulting from an altered low-affinity Ca 2+ binding site in the channel pore and thus causes malignant hyperthermia (MH). Currently, this condition has worldwide economic consequences and represents an economical major concern in the swine industry (D.H. MACLENNAN & al. [1]; M. FILL & al. [2]; Y. KIM & al. [3]). MH is a hypermetabolic syndrome involving the skeletal muscle characterized by muscle rigidity, hyperthermia, tachycardia, tachypnea, increased oxygen consumption, cyanosis, cardiac dysrhythmias, metabolic acidosis, respiratory acidosis, unstable arterial blood pressure, and death (E. SUSAN & al. [4]). The primary features of MH are a direct