Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces |
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| Institution: | 1. Biological Resources Center, KRIBB, Daejeon 305-806, Republic of Korea;2. Department of Biology, Chungnam National University, Daejeon 306-764, Republic of Korea;3. University of Science & Technology, 52 Eoeun-dong, Yuseong-gu, Daejeon, 305-333, Republic of Korea;4. Laboratory of Microbial Function, KRIBB, Daejeon 305-806, Republic of Korea;5. Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea;1. University of Cologne, Department of Terrestrial Ecology, Faculty of Zoology, Zülpicher Str. 47b, 50674 Köln, Germany;2. Leibniz-Centre for Agricultural Landscape Research ZALF, Institute of Landscape Biogeochemistry, Eberswalder Strasse 84, 15374 Müncheberg, Germany;1. Institute of Medical Microbiology and Hygiene, University of Tübingen, Tübingen, Germany;2. Department of Chemical Sciences, University of Naples Federico II, Naples, Italy;3. Institute of Radiology, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Tübingen, Germany;1. Institue of Environmental Biology, Ecology&Biodiversity, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands;2. Department of Plant Science, Laboratory of Nematology, Wageningen University, 6700 ES Wageningen, the Netherlands |
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| Abstract: | There have been an increasing number of reports of yeast systemic infection involving Saccharomyces cerevisiae strains. The development of a rapid and reliable diagnostic tool is therefore warranted in order to explore the distribution of S. cerevisiae as an opportunistic pathogen in humans. In this study, we designed and validated five primer sets targeting the 26S rRNA gene of S. cerevisiae and the S. sensu stricto complex using 26 yeast strains. Among them, two sets of primers specifically amplified the 26S rRNA gene and the ITS region of S. cerevisiae strains, and three sets were specific for amplifying the same genes in the S. sensu stricto complex. After determining the optimal conditions of two primer pairs for quantitative real time PCR, human fecal samples were analyzed to examine the distribution of S. cerevisiae and the S. sensu stricto complex. It was possible to detect a single cell of S. cerevisiae in environmental sample. Qualitative PCR revealed that out of eleven fecal samples tested, one sample contained S. cerevisiae and four samples contained the S. sensu stricto complex. Quantitative real time PCR revealed that the target gene copy numbers of S. cerevisiae and the S. sensu stricto complex were 0.84 and 2.44 respectively, in 1 ng of DNA from the bulk fecal community. |
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