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

• Published in: International journal of food microbiology 2014-05, Vol.178, p.107-112
• Main Authors: Ishikawa, Hiroshi, Kasahara, Kohei, Sato, Sumie, Shimakawa, Yasuhisa, Watanabe, Koichi
• Format: Article
• Language: English
• Subjects: Basidiomycota - genetics; Basidiomycota - physiology; Biological and medical sciences; Dairy product; Dairy Products - microbiology; DNA Primers - genetics; Filobasidiella neoformans; Food industries; Food microbiology; Food Microbiology - methods; Fundamental and applied biological sciences. Psychology; Loop-mediated isothermal amplification (LAMP); Nucleic Acid Amplification Techniques; Probiotics; Rapid detection; Rapid DNA extraction; rDNA internal transcribed spacer 2 (ITS2); Sensitivity and Specificity; Time
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/j.ijfoodmicro.2014.03.008

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 102cells/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 102cells/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 103cells/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 105cells/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. •A LAMP primer set targeting the ITS2 region of F. neoformans rDNA was developed.•LAMP was more sensitive than qPCR for the detection of crude F. neoformans DNA.•LAMP is suitable for the primary screening of food products for yeast contamination.