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Journal of Clinical Microbiology, October 2001, p. 3617-3622, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3617-3622.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Multiplex PCR Using Internal Transcribed Spacer 1 and 2 Regions for Rapid Detection and Identification of Yeast Strains

Shin-Ichi Fujita,1,2,* Yasuko Senda,2 Shigeki Nakaguchi,2 and Takuma Hashimoto1

Department of Laboratory Medicine, Graduate School of Medical Science,1 and Department of Clinical Laboratory, Kanazawa University Hospital,2 School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Japan

Received 12 February 2001/Returned for modification 6 March 2001/Accepted 22 July 2001

Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since Trichosporon asahi and T. asteroides showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as Candida guilliermondii, 2 as C. krusei, and 1 as C. zeylanoides). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).

* Corresponding author. Mailing address: Department of Laboratory Medicine, Graduate School of Medical Science, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Japan. Phone: (076) 265-2006. Fax: (076) 243-3276. E-mail: fujita-knz{at}umin.ac.jp.

Journal of Clinical Microbiology, October 2001, p. 3617-3622, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3617-3622.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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