Establishment of a Novel Molecular Detection Method for Sarcocystis in Venison

Recently, horse meat ( ) contaminated with spp. caused food poisoning in Japan. An official detection method provided by the Ministry of Health, Labour and Welfare (MHLW), Japan, was designed to detect to diagnose and control outbreaks of food poisoning. In 2011, -contaminated venison also caused food poisoning. However, the official MHLW detection method was not adequate for detecting spp. in venison. In this study, we established a novel PCR-based detection method that amplifies 18S rRNA gene based on the conserved region of the sequence in 32 species of for screening and quantification. Fifty venison samples from three areas in Hokkaido were examined by the MHLW method and the novel detection method. All samples were spp.-positive. A sequence analysis indicated the presence of a species of specific to sika deer ( ), and not to horses. Another primer pair was designed for a quantitative real-time PCR assay to determine the copy number of the Sarcocystis-18S rRNA gene in parasitized venison. The melting curve analysis revealed high specificity of this assay. The calculated curve demonstrated that this quantitative PCR assay showed value of 0.993 with 10-10 copies. Using this quantitative real-time PCR assay, the gene copy numbers were determined in 50 venison samples. The copy numbers of each sample ranged from 10 to 10 per gram. The copy numbers differed according to the area in Hokkaido. This indicates that the density of spp. that infect Sika deer in Hokkaido is affected by the area. The novel screening and quantitative PCR method for in venison was useful for collecting epidemiological information on in wild Japanese sika deer, which will contribute to improve the safety of venison products in Japan.