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• W2054091657 abstract "Background: Invasive Aspergillosis causes significant morbidity and mortality in bone marrow transplantation recipients. In the absence of rapid, sensitive, specific, and quantitative diagnostic assays, it is estimated that> 90% of undiagnosed and untreated cases result in death. Here we report a rapid, sensitive, and specific real time PCR assay to diagnose Aspergillus infections. Methods: We set out to develop a real-time PCR method to detect and quantify 8 medically important Aspergillus species (A. fumigatus, A. clavatus, A. flavus, A. glaucus, A. nidulans, A. niger, A. terreus, and A. versicolor) in a single assay. We carefully aligned several Aspergillus multicopy rRNA genes to search for a target that is both specific for Aspergillus and conserved for all 8 species. To evaluate the diagnostic utility of a potential target, we performed real-time PCR on blood specimens that were spiked with serially diluted conidia from each of these 8 species. Additionally, we evaluated the effects of DNA extraction yield on the assay’s sensitivity. For this purpose, we evaluated 3 commercially available DNA extraction kits: EZNA, Qiagen, and MagNA Pure DNA extraction methods. Results: Through sequence analysis, we designed a primer pair and TaqMan probe specific for a region of the 18s rRNA gene that is conserved for all 8 species. On application, our results demonstrate that this assay can detect and quantify as few as 100 copies of all 8 medically significant Aspergillus conidia per mL of spiked blood. In evaluating 3 commercially available DNA extraction kits, we found that the EZNA kit provided the maximum DNA yield and increased assay sensitivity. Moreover, we determined that mechanical disruption of the specimen by glass beads resulted in higher DNA yield and increased assay sensitivity. Conclusion: Developing a single molecular diagnostic assay for all medically important Aspergillus species is major challenge. Currently we are performing both retrospective and prospective studies to evaluate its diagnostic utility. Recently, the galactomannan-based assay showed promise in diagnosis of invasive aspergillosis; however, it still has high false-positivity and false-negativity. Furthermore, an optimum OD cutoff value has yet to be established for early diagnosis. Therefore, real-time PCR assay can provide an alternative means of diagnosis of invasive aspergillosis. Background: Invasive Aspergillosis causes significant morbidity and mortality in bone marrow transplantation recipients. In the absence of rapid, sensitive, specific, and quantitative diagnostic assays, it is estimated that> 90% of undiagnosed and untreated cases result in death. Here we report a rapid, sensitive, and specific real time PCR assay to diagnose Aspergillus infections. Methods: We set out to develop a real-time PCR method to detect and quantify 8 medically important Aspergillus species (A. fumigatus, A. clavatus, A. flavus, A. glaucus, A. nidulans, A. niger, A. terreus, and A. versicolor) in a single assay. We carefully aligned several Aspergillus multicopy rRNA genes to search for a target that is both specific for Aspergillus and conserved for all 8 species. To evaluate the diagnostic utility of a potential target, we performed real-time PCR on blood specimens that were spiked with serially diluted conidia from each of these 8 species. Additionally, we evaluated the effects of DNA extraction yield on the assay’s sensitivity. For this purpose, we evaluated 3 commercially available DNA extraction kits: EZNA, Qiagen, and MagNA Pure DNA extraction methods. Results: Through sequence analysis, we designed a primer pair and TaqMan probe specific for a region of the 18s rRNA gene that is conserved for all 8 species. On application, our results demonstrate that this assay can detect and quantify as few as 100 copies of all 8 medically significant Aspergillus conidia per mL of spiked blood. In evaluating 3 commercially available DNA extraction kits, we found that the EZNA kit provided the maximum DNA yield and increased assay sensitivity. Moreover, we determined that mechanical disruption of the specimen by glass beads resulted in higher DNA yield and increased assay sensitivity. Conclusion: Developing a single molecular diagnostic assay for all medically important Aspergillus species is major challenge. Currently we are performing both retrospective and prospective studies to evaluate its diagnostic utility. Recently, the galactomannan-based assay showed promise in diagnosis of invasive aspergillosis; however, it still has high false-positivity and false-negativity. Furthermore, an optimum OD cutoff value has yet to be established for early diagnosis. Therefore, real-time PCR assay can provide an alternative means of diagnosis of invasive aspergillosis." @default.
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• W2054091657 date "2005-02-01" @default.
• W2054091657 modified "2023-09-25" @default.
• W2054091657 title "Development of a real-time PCR assay to diagnose invasive Aspergillosis" @default.
• W2054091657 doi "https://doi.org/10.1016/j.bbmt.2004.12.218" @default.
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