Development of a multiplex-PCR assay for differentiation of Cryptococcus species using the PRP8 gene region

Cryptococcus is an opportunistic pathogen responsible for cryptococcosis, a fungal infection with high mortality, particularly in immunocompromised individuals. Accurate and rapid species identification is critical for effective diagnosis and treatment. This study developed and standardized a multiplex PCR protocol targeting the polymorphic PRP8 gene, a novel molecular marker proposed for differentiation of C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. DNA was extracted from reference strains and primers were manually designed. The protocol demonstrated high specificity, with no amplification observed in negative controls, and reproducibility under optimized conditions, including an annealing temperature of 52 °C and 3% DMSO. This method is cost-effective, time-efficient, and robust, making it suitable for laboratories with limited resources. Compared to traditional targets such as coat genes and ribosomal RNA, the PRP8 gene offers enhanced resolution due to its intronic polymorphisms, allowing precise differentiation of molecular types. This innovation provides a valuable tool for clinical diagnostics and epidemiological studies, facilitating the understanding of geographic distribution, infection patterns and antifungal resistance profiles.