Detection and identiﬁcation of probable endemic fungal pathogen, Cryptococcus gattii, and worldwide pathogen, Cryptococcus neoformans, by real-time PCR
Kazuo Satoh1,2, Mari Maeda1, Yoshiko Umeda1, Yoshiharu Miyajima3 and Koichi Makimura1,4
1Teikyo University Institute of Medical Mycology
2The Association for Preventive Medicine of Japan, Emachu Kinshi-cho Bldg, 1-19-10 Mori, Koto-ku, Tokyo 135-0001
3Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
4Genome Research Center, Graduate School of Medicine and Faculty of Medicine, Teikyo University, 359 Otsuka, Hachioji, Tokyo 192-0395
Areal-time PCR method for detection and identiﬁcation of Cryptococcus neoformans and Cryptococcus gattii was developed and evaluated using DNA from single-colony or koala nasal smears. Two TaqMan minor groove binder probes that distinguished between these species were designed corresponding to the internal sequences of the CAP59 gene for both species. The real-time PCR assay had 100% speciﬁcity, as assessed using 13 reference strains and 300 environmental strains. Twelve smear samples from healthy koalas were analyzed by direct real-time PCR. This method successfully detected C. gattii and C. neoformans in one and three koalas, respectively.