Multiplex PCR using internal transcribed spacer 1 and 2 regions for rapid detection and identification of yeast strains

Abstract

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).

FIG. 1

Schematic representation of the fungal ribosomal genes containing the primer target areas used in this study.

FIG. 2

Gels showing amplification by PCR and length variations in the ITS regions of medically important Candida isolates. Lane 1, C. albicans; lane 2, C. dubliniensis; lane 3, C. tropicalis; lane 4, C. parapsilosis; lane 5, C. guilliermondii; lane 6, C. krusei; lane 7, C. glabrata; lane 8, C. lusitaniae; lanes M, 100-bp DNA ladder.

FIG. 3

ME patterns of C. albicans IFO 0745 (A), C. tropicalis IFO 1400 (B), and C. guilliermondii IFO 0566 (C). Arrows indicate DNA size markers of 100 bp (left) and 800 bp (right), and asterisks indicate PCR products (A, 341 and 531 bp; B, 329 and 518 bp; and C, 380 and 602 bp). Int., intensity.