Purchase this article with an account.
Lisa Kercher, Stephanie A. Wardwell, Kirk R. Wilhelmus, Bradley M. Mitchell; Molecular Screening of Donor Corneas for Fungi before Excision. Invest. Ophthalmol. Vis. Sci. 2001;42(11):2578-2583.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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
103 genome equivalents, when using genomic DNA as a
template and 102 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.
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
This PDF is available to Subscribers Only