title: Development and evaluation of a real-time quantitative PCR assay for detection and enumeration of yeasts of public health interest in dairy products.
abstract: Yeast contamination is a problem in the food industry as a cause of spoilage. Moreover, various species of yeasts are known to be capable of causing opportunistic infections in humans. We have developed a real-time quantitative PCR (qPCR) assay to directly detect and quantify nine emerging opportunistic yeast species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Filobasidiella neoformans, Issatchenkia orientalis, Trichosporon asahii, and Trichosporon jirovecii) in dairy product samples. We designed six primer pairs, conserved sequences of the variable D1/D2 domains of the 26S rRNA gene, to detect the yeasts and demonstrated their specificity. The qPCR assay could accurately quantify emerging opportunistic yeasts in an artificially contaminated dairy product. qPCR with the primer pairs we designed, was very sensitive and will allow producers to enumerate contaminating yeasts and identify whether they are opportunistic pathogens, in only 4 to 5h. This assay can easily be extended to other food items and to a variety of food-monitoring initiatives.
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. hiroshi.makino@yher.be
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Makino
authors.forename: Junji
authors.initials: J
authors.lastname: Fujimoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Watanabe
mesh_terms: D015169:Colony Count, Microbial; D017931:DNA Primers / Q000235:genetics; D003611:Dairy Products / Q000382:microbiology*; D005516:Food Microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors; D015003:Yeasts / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S