title: Simple and rapid method for the detection of Filobasidiella neoformans in a probiotic dairy product by using loop-mediated isothermal amplification.
abstract: Yeast contamination is a serious problem in the food industry and a major cause of food spoilage. Several yeasts, such as Filobasidiella neoformans, which cause cryptococcosis in humans, are also opportunistic pathogens, so a simple and rapid method for monitoring yeast contamination in food is essential. Here, we developed a simple and rapid method that utilizes loop-mediated isothermal amplification (LAMP) for the detection of F. neoformans. A set of five specific LAMP primers was designed that targeted the 5.8S-26S rDNA internal transcribed spacer 2 region of F. neoformans, and the primer set's specificity was confirmed. In a pure culture of F. neoformans, the LAMP assay had a lower sensitivity threshold of 10(2)cells/mL at a runtime of 60min. In a probiotic dairy product artificially contaminated with F. neoformans, the LAMP assay also had a lower sensitivity threshold of 10(2)cells/mL, which was comparable to the sensitivity of a quantitative PCR (qPCR) assay. We also developed a simple two-step method for the extraction of DNA from a probiotic dairy product that can be performed within 15min. This method involves initial protease treatment of the test sample at 45°C for 3min followed by boiling at 100°C for 5min under alkaline conditions. In a probiotic dairy product artificially contaminated with F. neoformans, analysis by means of our novel DNA extraction method followed by LAMP with our specific primer set had a lower sensitivity threshold of 10(3)cells/mL at a runtime of 60min. In contrast, use of our novel method of DNA extraction followed by qPCR assay had a lower sensitivity threshold of only 10(5)cells/mL at a runtime of 3 to 4h. Therefore, unlike the PCR assay, our LAMP assay can be used to quickly evaluate yeast contamination and is sensitive even for crude samples containing bacteria or background impurities. Our study provides a powerful tool for the primary screening of large numbers of food samples for yeast contamination.
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. Electronic address: h-ishikawa@yakult.co.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ishikawa
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Kasahara
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan.
authors.forename: Sumie
authors.initials: S
authors.lastname: Sato
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Shimakawa
authors.affiliation: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. Electronic address: koichi-watanabe@yakult.co.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Watanabe
mesh_terms: D001487:Basidiomycota / Q000235:genetics / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D003611:Dairy Products / Q000382:microbiology*; D005516:Food Microbiology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D019936:Probiotics; D012680:Sensitivity and Specificity; D013995:Time
chemical_list: D017931:DNA Primers
keywords: Dairy product; Filobasidiella neoformans; Loop-mediated isothermal amplification (LAMP); Rapid DNA extraction; Rapid detection; rDNA internal transcribed spacer 2 (ITS2)