Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway
Background: Prior studies illustrate the presence and clinical importance of detecting <i>Aspergillus</i> species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (...
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MDPI AG
2020-04-01
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author | Tuang Yeow Poh Nur A’tikah Binte Mohamed Ali Louisa L.Y. Chan Pei Yee Tiew Sanjay H. Chotirmall |
author_facet | Tuang Yeow Poh Nur A’tikah Binte Mohamed Ali Louisa L.Y. Chan Pei Yee Tiew Sanjay H. Chotirmall |
author_sort | Tuang Yeow Poh |
collection | DOAJ |
description | Background: Prior studies illustrate the presence and clinical importance of detecting <i>Aspergillus</i> species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of <i>Aspergillus</i> in specimens from the human airway. Droplet digital PCR (ddPCR) however, presents an alternative methodology allowing higher sensitivity and accuracy of such quantification but remains to be evaluated in head-to-head fashion using specimens from the human airway. Here, we implement a standard duplex TaqMan PCR protocol, and assess if ddPCR is superior in quantifying airway <i>Aspergillus</i> when compared to standard qPCR. Methods: The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in <i>n</i> = 20 sputum specimens obtained from non-diseased (<i>n</i> = 4), chronic obstructive pulmonary disease (COPD; <i>n</i> = 8) and non-cystic fibrosis bronchiectasis (<i>n</i> = 8) patients where <i>Aspergillus</i> status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed. Results: Standard qPCR and ddPCR were both able to detect, even at low abundance, <i>Aspergillus</i> species (<i>Aspergillus fumigatus - A. fumigatus</i> and <i>Aspergillus terreus - A. terreus</i>) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of <i>A. terreus</i> particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR. Conclusion: ddPCR has greater sensitivity for <i>A. terreus</i> detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of <i>A. terreus</i> species in chronic respiratory disease states such as bronchiectasis. |
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language | English |
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spelling | doaj.art-a1860557b0ff493aa3734179eba1c6cd2023-11-19T22:44:39ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-04-01219304310.3390/ijms21093043Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human AirwayTuang Yeow Poh0Nur A’tikah Binte Mohamed Ali1Louisa L.Y. Chan2Pei Yee Tiew3Sanjay H. Chotirmall4Translational Respiratory Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232, SingaporeTranslational Respiratory Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232, SingaporeTranslational Respiratory Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232, SingaporeTranslational Respiratory Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232, SingaporeTranslational Respiratory Research Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232, SingaporeBackground: Prior studies illustrate the presence and clinical importance of detecting <i>Aspergillus</i> species in the airways of patients with chronic respiratory disease. Despite this, a low fungal biomass and the presence of PCR inhibitors limits the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of <i>Aspergillus</i> in specimens from the human airway. Droplet digital PCR (ddPCR) however, presents an alternative methodology allowing higher sensitivity and accuracy of such quantification but remains to be evaluated in head-to-head fashion using specimens from the human airway. Here, we implement a standard duplex TaqMan PCR protocol, and assess if ddPCR is superior in quantifying airway <i>Aspergillus</i> when compared to standard qPCR. Methods: The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in <i>n</i> = 20 sputum specimens obtained from non-diseased (<i>n</i> = 4), chronic obstructive pulmonary disease (COPD; <i>n</i> = 8) and non-cystic fibrosis bronchiectasis (<i>n</i> = 8) patients where <i>Aspergillus</i> status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed. Results: Standard qPCR and ddPCR were both able to detect, even at low abundance, <i>Aspergillus</i> species (<i>Aspergillus fumigatus - A. fumigatus</i> and <i>Aspergillus terreus - A. terreus</i>) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of <i>A. terreus</i> particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR. Conclusion: ddPCR has greater sensitivity for <i>A. terreus</i> detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of <i>A. terreus</i> species in chronic respiratory disease states such as bronchiectasis.https://www.mdpi.com/1422-0067/21/9/3043<i>Aspergillus fumigatus</i><i>Aspergillus terreus</i>qPCRddPCRquantification |
spellingShingle | Tuang Yeow Poh Nur A’tikah Binte Mohamed Ali Louisa L.Y. Chan Pei Yee Tiew Sanjay H. Chotirmall Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway International Journal of Molecular Sciences <i>Aspergillus fumigatus</i> <i>Aspergillus terreus</i> qPCR ddPCR quantification |
title | Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway |
title_full | Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway |
title_fullStr | Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway |
title_full_unstemmed | Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway |
title_short | Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of <i>Aspergillus</i> species in the Human Airway |
title_sort | evaluation of droplet digital polymerase chain reaction ddpcr for the absolute quantification of i aspergillus i species in the human airway |
topic | <i>Aspergillus fumigatus</i> <i>Aspergillus terreus</i> qPCR ddPCR quantification |
url | https://www.mdpi.com/1422-0067/21/9/3043 |
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