Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces.
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.
Chang
Ho-Won
HW
Biological Resources Center, KRIBB, Daejeon 305-806, Republic of Korea.
Nam
Young-Do
YD
Sung
Youlboong
Y
Kim
Kyoung-Ho
KH
Roh
Seong Woon
SW
Yoon
Jung-Hoon
JH
An
Kwang-Guk
KG
Bae
Jin-Woo
JW
D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis*; D005243:Feces / Q000382:microbiology; D006801:Humans; D016533:Mycological Typing Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000032:analysis*; D012440:Saccharomyces / Q000145:classification / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S