New PCR primer pairs specific for Cryptococcus neoformans serotype A or B prepared on the basis of random amplified polymorphic DNA fingerprint pattern analyses

Abstract

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respective C. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

FIG. 1

RAPD fingerprint patterns of seven strains of C. neoformans serotype A isolated in Brazil obtained with primer R-1. Lane M, DNA ladder marker used as the molecular size standard; lanes 1 to 7, clinical serotype A isolates from Brazil.

FIG. 2

RAPD fingerprint patterns of the reference C. neoformans serotype A, B, C, D and AD strains obtained with primer R-2. Lane M, marker; lanes A to AD, reference serotype A, B, C, D, and AD strains, respectively. The reference strains were CDC 551 (serotype A), NIH 112 (serotype B), NIH 18 (serotype C), IFM 5857 (serotype D), and LY 23 (serotype AD). The arrows indicate the DNA bands that were obtained by RAPD analysis and that were extracted and sequenced in the present experiment.

FIG. 3

Newly determined sequence of 695-bp band of DNA from a C. neoformans serotype A strain obtained by RAPD analysis and the sequences (underlined) of the new PCR primer pair that was prepared. Newly determined sequence of 448-bp band of DNA from a C. neoformans serotype B strain obtained by RAPD analysis and the sequences (underlined) of the new PCR primer pair that was prepared.

FIG. 4

PCR identification of C. neoformans serotype A strains with the CNa-70-S and CNa-70-A PCR primer pair. Lane M, marker; lanes A to AD, reference strains of serotypes A, B, C, D, and AD, respectively; lanes CA, clinical serotype A isolates (CAMP-1, CAMP-2, and CAMP-4 from left to right, respectively) from Brazil.

FIG. 5

PCR identification of C. neoformans serotype B strains with the serotype A-specific CNa-29-S and CNa-70-A PCR primer pair. Serotype A and B strains were identified by the presence of amplified bands of 666 bp (along with one of 460 bp) and 290 bp, respectively. Lane M, marker; lanes A and B, reference strains of serotypes A (CDC 551) and B (NIH 112), respectively; lanes CA, clinical serotype A isolates (CAMP-1, CAMP-2, and CAMP-4 from left to right, respectively) from Brazil.

FIG. 6

PCR identification of two clinical strains of C. neoformans serotype B with the CNb-49-S and CNb-49-A PCR primer pair. Lane M, marker; lanes A and B, reference strains of serotypes A (CDC 551) and B (NIH 112), respectively; lanes CB, serotype B clinical isolates (CAMP-3 and CAMP-24 on the left and right, respectively) from Brazil.