GenotypingCryptococcus neoformansvar. neoformans with specific primers designed from PCR-fingerprinting bands sequenced using a modified PCR-based strategy

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Abstract
Cryptococcus neoformans var. neoformans can be subdivided into six genotypes (VN1-VN6) based on different combinations of four major bands (420, 475, 540 and 800 bp) obtained by polymerase chain reaction (PCR) fingerprinting using the (GACA)4 primer. The aim of this study was to identify specific primers able to amplify these bands. A modified PCR-based sequencing strategy was adopted to overcome the limitations of using (GACA)4 as a single cycle sequencing primer. The original bands, made up of amplicons with two terminal (GACA)4 sequences, were digested with five restriction enzymes. Digestion products yielding two or three fragments were selected. Each fragment was expected to have no more than one terminal (GACA)4 sequence, making cycle sequencing possible. Fragments were purified and sequenced with the (GACA)4 primer. New primers specific for each of the four major bands were then designed and the remaining regions were sequenced using both purified bands and PCR-fingerprinting products as template. These primers were used to amplify the genomic DNA of 12 C. neoformans strains and five strains of other yeast species. The new primers, used as separate pairs or in a mixture of all pairs, amplified the expected bands only in C. neoformans var. neoformans strains, confirming the species specificity of the bands selected for molecular typing of this yeast.

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