RAPID IDENTIFICATION OF THE VARIETIES AND GENOTYPES OF CRYPTOCOCCUS NEOFORMANS SPECIES COMPLEX USING A HIGH-THROUGHPUT FLOW CYTOMETER

摘要

Nucleic acid probes and molecular method to identify the varieties and genotypic groups within C. neoformans species complex. The method employs a flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin-R-phycoerythrin. The assay is specific and sensitive, and allows discrimination of 1bp mismatch with no apparent cross-reactivity and is capable of detecting 101 to 103 genome copies. The assay can be used directly with yeast cells or isolated DNA, can be undertaken in less than one hour following PCR amplification and permits identification of species in a multiplex format. In addition, to multiplex capability, the assay allows simultaneous detection of target sequences in a single reaction.