A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tis
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A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue Vishal Gupta Æ Ronald R. Cobb Æ Lauren Brown Æ Laraine Fleming Æ Nilay Mukherjee
Received: 21 June 2007 / Accepted: 6 November 2007 / Published online: 23 November 2007 Ó Springer Science+Business Media B.V. 2007
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 V. Gupta Department of Bioengineering, Rice University, 6100 Main St., Houston, TX 77005, USA R. R. Cobb L. Brown L. Fleming N. Mukherjee (&) Regeneration Technologies, Inc., 11621 Research Circle, P.O. Box 2650, Alachua, FL 32616-2650, USA e-mail: [email protected]
the time required to identify fungal contamination in tissue implants. Keywords Quantitative polymerase chain reaction Organ transplant Fungal organisms Donor tissues Limit of detection
Introduction The use of soft tissue grafting techniques, such as osteochondral grafts, has been growing rapidly over the past several years. The use of autologous grafts still remains the tissue of choice in most procedures mainly due to the lack of immune rejection. However, there still remains the issue of donor site availability and the tedious procedure associated with collection of tissue from the donor site. Due to these potential limitations, the use of allograft tissues has continued to grow. Recent reports describing the risk of contamination of allografts have forced some tissue banks to implement various cleansing procedures (Eastlund 2006; Galvan et al. 2006). Recent advances in donor screening, bacterial and viral testing and aseptic processing have reduced the risk of disease transmission and virtually eliminated reports of contaminated grafts (Kainer et al. 2004). However, there are recent reports warning clinicians of the risk of allograft material (Conrad et al. 1995; Eggen and Norbdo 1992; Kainer et al. 2004; Simonds et al. 1992; Vangsness et al
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