Kasahara Kohei, Ishikawa Hiroshi, Sato Sumie, Shimakawa Yasuhisa, Watanabe Koichi
Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
FEMS Microbiol Lett. 2014 Aug;357(2):208-16. doi: 10.1111/1574-6968.12512. Epub 2014 Jul 14.
Rapid detection of yeast contamination is important in the food industry. We have developed loop-mediated isothermal amplification (LAMP) assays to detect the emerging opportunistic pathogenic yeasts: Candida albicans, Candida glabrata, Candida tropicalis, the Candida parapsilosis group, Trichosporon asahii, and Trichosporon mucoides. These yeasts may cause deep-seated candidiasis or trichosporonosis. Four LAMP primer sets specific for Candida were designed to target the internal transcribed spacer 2 (ITS2) region between the 5.8S and 26S rRNA genes, and two LAMP primer sets specific for Trichosporon were designed to target the intergenic spacer 1 (IGS1) region between the 26S and 5S rRNA genes. The LAMP assays could detect these yeasts in a range between 10(0) and 10(3) cells mL(-1) in a contaminated dairy product within 1 h. We also developed multiplex LAMP assays to detect these Candida or Trichosporon species in a single reaction. Multiplex LAMP assays can detect contamination if at least one of the target species is present; they are more time- and cost-efficient than conventional methods and could detect target yeasts with sensitivity close to that of the LAMP assays. Multiplex LAMP assays established in this study can be used as a primary screening method for yeast contamination in food products.
快速检测酵母污染在食品工业中至关重要。我们开发了环介导等温扩增（LAMP）检测方法，以检测新出现的机会致病性酵母：白色念珠菌、光滑念珠菌、热带念珠菌、近平滑念珠菌属、阿萨希毛孢子菌和黏液毛孢子菌。这些酵母可能会引起深部念珠菌病或毛孢子菌病。设计了四组针对念珠菌的LAMP引物，靶向5.8S和26S rRNA基因之间的内部转录间隔区2（ITS2）区域，设计了两组针对毛孢子菌的LAMP引物，靶向26S和5S rRNA基因之间的基因间隔区1（IGS1）区域。LAMP检测方法能够在1小时内检测出受污染乳制品中细胞浓度在10(0)至10(3) 个/mL之间的这些酵母。我们还开发了多重LAMP检测方法，以在单一反应中检测这些念珠菌或毛孢子菌物种。如果至少存在一种目标物种，多重LAMP检测方法就能检测出污染情况；它们比传统方法更节省时间和成本，并且能够以接近LAMP检测方法的灵敏度检测目标酵母。本研究中建立的多重LAMP检测方法可作为食品中酵母污染的初步筛选方法。
