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 (...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-04-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/21/9/3043 |
| Summary: | 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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| ISSN: | 1661-6596 1422-0067 |