PURPOSE: To develop panfungal and Candida albicans species-specific polymerase chain reaction (PCR) assays to screen donor eyes for fungal contamination before corneal excision. METHODS: PCR primers were designed for either the broad-spectrum detection of fungal DNA or the specific detection of C. albicans DNA. Their sequences were based on rDNA regions highly conserved among and specific to fungi and C. albicans, respectively. PCR conditions with the two primer sets were optimized and tested for sensitivity using purified C. albicans genomic DNA and a plasmid containing the relevant region of C. albicans DNA. The specificity of the primer sets was established using higher eukaryotic, fungal, prokaryotic, and viral DNAs as PCR templates. Donor eye swab specimens were collected before corneal excision. DNA was extracted from the specimens and tested by both PCR assays. RESULTS: The lower limit of detection for both primer sets was consistently 10(3) genome equivalents, when using genomic DNA as a template and 10(2) copies of plasmid. The fungal PCR assay amplified DNA from all fungal species tested but did not amplify any of the selected mammalian, bacterial, or viral DNA. The C. albicans PCR detected the C. albicans DNA but was negative for all other DNA substrates, including the other fungal templates. Thirty-five percent of the donor eye samples tested were positive for fungus, and 19% were positive for C. albicans DNA. CONCLUSIONS: The PCR assays allowed the rapid screening of DNA extracted from specimens collected from corneal donors for potential fungal contamination. The assay was highly sensitive and specific for screening corneal surfaces. The results suggest that approximately one-third of donor eyes tested harbor fungi on the ocular surface.
PURPOSE: To develop panfungal and Candida albicans species-specific polymerase chain reaction (PCR) assays to screen donor eyes for fungal contamination before corneal excision. METHODS: PCR primers were designed for either the broad-spectrum detection of fungal DNA or the specific detection of C. albicans DNA. Their sequences were based on rDNA regions highly conserved among and specific to fungi and C. albicans, respectively. PCR conditions with the two primer sets were optimized and tested for sensitivity using purified C. albicans genomic DNA and a plasmid containing the relevant region of C. albicans DNA. The specificity of the primer sets was established using higher eukaryotic, fungal, prokaryotic, and viral DNAs as PCR templates. Donor eye swab specimens were collected before corneal excision. DNA was extracted from the specimens and tested by both PCR assays. RESULTS: The lower limit of detection for both primer sets was consistently 10(3) genome equivalents, when using genomic DNA as a template and 10(2) copies of plasmid. The fungal PCR assay amplified DNA from all fungal species tested but did not amplify any of the selected mammalian, bacterial, or viral DNA. The C. albicans PCR detected the C. albicans DNA but was negative for all other DNA substrates, including the other fungal templates. Thirty-five percent of the donor eye samples tested were positive for fungus, and 19% were positive for C. albicans DNA. CONCLUSIONS: The PCR assays allowed the rapid screening of DNA extracted from specimens collected from corneal donors for potential fungal contamination. The assay was highly sensitive and specific for screening corneal surfaces. The results suggest that approximately one-third of donor eyes tested harbor fungi on the ocular surface.
Authors: Xia Hua; Xiaoyong Yuan; Bradley M Mitchell; Michael C Lorenz; Denis M O'Day; Kirk R Wilhelmus Journal: Mol Vis Date: 2009-07-30 Impact factor: 2.367