Development of a singleplex PCR assay for rapid identification and differentiation of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, Cryptococcus gattii, and hybrids

Abstract

A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and large-scale epidemiologic studies.

Fig 1

Multiple sequence alignment of fragments of the STR1 gene orthologs in type strains. Primers STR1F and STR1R are underlined. Two adjacent introns within the gene fragment are marked by a gray background. Gaps representing intron loss are indicated by dashes.

Fig 2

Neighbor-joining tree for type strains, including hybrid strains obtained by analysis of 170-bp fragments of the STR1 gene. Bootstrap values (500 replicates) are indicated for the main branches. Intron is excluded from the phylogenetic analysis.

Fig 3

Agarose gel electrophoresis of PCR products. Species, variety and major genotype are depicted in brackets. Lanes: M, DL2000 ladder; 1, WM148 (C. neoformans var. grubii VNI); 2, Bt60 (C. neoformans var. grubii VNB); 3, WM626 (C. neoformans var. grubii VNII); 4, WM628 (C. neoformans AD hybrid VNIII); 5, WM629 (C. neoformans var. neoformans VNIV); 6, WM179 (C. gattii VGI); 7, WM178 (C. gattii VGII); 8, WM161 (C. gattii VGIII); 9, WM779 (C. gattii VGIV); 10, CBS10488 (C. neoformans×C. gattii BD hybrid); 11, CBS10496 (C. neoformans × C. gattii AB hybrid); 12, negative control.