Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex

Abstract

The Cryptococcus species complex contains two closely related basidiomycetous yeasts: Cryptococcus neoformans and C. gattii, which cause cryptococcosis in humans and other animals. The species and varieties are characterized, by different clinical, epidemiological, biochemical and molecular features. The currently used identification methods are either time-consuming or not anymore commercially available. However, a rapid, sensitive and robust assay for the detection of these pathogens is vital for early diagnosis and appropriate treatment decisions. To overcome those limitations, four padlock probes targeting speciesspecific single nucleotide polymorphisms at the internal transcribed spacers (ITSs) of the RNA gene locus were developed and applied during isothermal hyperbranched rolling circle amplification (HRCA). The probes were tested against 99 samples, including 94 clinical cryptococcal cultures, three closely related Cryptococcus species, and two clinical specimens. The use of the padlock probes and the combination of probe signal amplification by HRCA provided a quick and sensitive assay for the accurate identification of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. HRCA was also useful to detect hybrids, when they were heterozygous at the ITS locus. The HRCA results were in agreement with previous genotyping data based on PCR fingerprinting, amplified fragment length polymorphism and ITS sequencing.

Development of a Singleplex PCR Assay for Rapid Identification and Differentiation of Cryptococcus neoformans var.grubii, Cryptococcus neoformans var. neoformans, Cryptococcus gattii, and Hybrids

“…neoformans and C. neoformans var. grubii, respectively, by the PCR analysis, which was in correspondence with other reports using single or a few target genes (3,10,11). This result may be due to genomic instability in AD hybrid strains with loss of heterozygosity for the allele of STR1 gene, which may be resolved by molecular tools designed for use at the whole-genome level, such as amplified fragment length polymorphism (AFLP) and PCR fingerprinting (2,7,19).…”

“…Several molecular approaches, such as multiplex PCR, PCRrestriction fragment length polymorphism (RFLP) analysis, hyperbranched rolling circle amplification (HRCA), loop-mediated isothermal DNA amplification, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), have been developed to identify species, varieties, and hybrids (2,(8)(9)(10)(11)(12). Overall, these methods, while reliable, usually require expensive instruments or complicated methods.…”

Cryptococcus gattii Infections

“…Isothermal amplification approaches, including hyperbranched rolling-circle amplification (RCA), targeting single nucleotide polymorphisms (SNPs) in the ITS region, have also successfully identified C. gattii (347). This technology uses circularizing padlock probes of approximately a 50-to 100-mer length that contain target complementary sequences by a linker.…”

Identification ofCryptococcus gattiiby Use ofl-Canavanine Glycine Bromothymol Blue Medium and DNA Sequencing

“…The second approach is a molecular method that utilizes DNA sequencing of the D2 region of the fungal 28S large ribosomal subunit to distinguish C. neoformans from C. gattii. Others have reported differentiation of C. neoformans and C. gattii by the use of sequencing of the internal transcribed spacer region (3,6,7), but to our knowledge, this is the first report of the use of D2 large ribosomal subunit region sequencing for the identification of C. gattii.…”