A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue |
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Authors: | Vishal Gupta Ronald R Cobb Lauren Brown Laraine Fleming Nilay Mukherjee |
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Institution: | (1) Department of Bioengineering, Rice University, 6100 Main St., Houston, TX 77005, USA;(2) Regeneration Technologies, Inc., 11621 Research Circle, P.O. Box 2650, Alachua, FL 32616-2650, USA |
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Abstract: | To complement donor selection and tissue processing, rapid and reliable detection, discrimination, and quantification of fungal
pathogens are extremely important for tissues destined to be implanted into humans. The current detection method for fungal
pathogens, in particular, is difficult and time-consuming. Quantitative polymerase chain reaction (qPCR) technology is considered
one of the most sensitive methods to detect low levels of DNA. Here a qPCR method is described that can detect clinically
relevant, pathogenic fungal organisms. The assay allowed the quantification of fungal organisms within a tissue implant and
provides a means to identify the contaminating species. The primers for the qPCR assay were designed to amplify a conserved
region of the L2 region of the large ribosomal subunit (LSU) gene. This set of primers was able to detect fewer than 10 colony
forming units from Aspergillus and Candida species in spiked samples. Clinical samples were also evaluated using this method
and the data compared positively to the existing accepted 28-day fungal culture method for fungal detection. The qPCR method
described herein significantly reduced the time required to identify fungal contamination in tissue implants. |
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Keywords: | Quantitative polymerase chain reaction Organ transplant Fungal organisms Donor tissues Limit of detection |
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