Development of multiplex loop-mediated isothermal amplification assays to detect medically important yeasts in dairy products.

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.

Kasahara K ，Ishikawa H ，Sato S ，Shimakawa Y ，Watanabe K ... -  《-》

Development and evaluation of a real-time quantitative PCR assay for detection and enumeration of yeasts of public health interest in dairy products.

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.

Makino H ，Fujimoto J ，Watanabe K 《-》

Simple and rapid method for the detection of Filobasidiella neoformans in a probiotic dairy product by using loop-mediated isothermal amplification.

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.

Ishikawa H ，Kasahara K ，Sato S ，Shimakawa Y ，Watanabe K ... -  《-》

Comparison of a capillary gel electrophoresis-based multiplex PCR assay and ribosomal intergenic transcribed spacer-2 amplicon sequencing for identification of clinically important Candida species.

The performance of a commercially available Seegene Seeplex STI Master Panel 3 multiplex PCR for Candida species identification was compared with an internal transcribed spacer 2 (ITS2) PCR assay. We found that the Seeplex assay was specific for identification of C. albicans, C. krusei, C. parapsilosis, C. glabrata, C. tropicalis and C. dubliniensis.

Monstein HJ ，Tärnberg M ，Persis S ，Johansson AG ... -  《-》

Identification of Trichosporon asahii by PCR based on sequences of the internal transcribed spacer regions.

Sugita T ，Nishikawa A ，Shinoda T 《-》