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 (...

Full description

Bibliographic Details
Main Authors: Tuang Yeow Poh, Nur A’tikah Binte Mohamed Ali, Louisa L.Y. Chan, Pei Yee Tiew, Sanjay H. Chotirmall
Format: Article
Language:English
Published: MDPI AG 2020-04-01
Series:International Journal of Molecular Sciences
Subjects:
Online Access:https://www.mdpi.com/1422-0067/21/9/3043
_version_ 1797569585343365120
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.
first_indexed 2024-03-10T20:13:47Z
format Article
id doaj.art-a1860557b0ff493aa3734179eba1c6cd
institution Directory Open Access Journal
issn 1661-6596
1422-0067
language English
last_indexed 2024-03-10T20:13:47Z
publishDate 2020-04-01
publisher MDPI AG
record_format Article
series International Journal of Molecular Sciences
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
work_keys_str_mv AT tuangyeowpoh evaluationofdropletdigitalpolymerasechainreactionddpcrfortheabsolutequantificationofiaspergillusispeciesinthehumanairway
AT nuratikahbintemohamedali evaluationofdropletdigitalpolymerasechainreactionddpcrfortheabsolutequantificationofiaspergillusispeciesinthehumanairway
AT louisalychan evaluationofdropletdigitalpolymerasechainreactionddpcrfortheabsolutequantificationofiaspergillusispeciesinthehumanairway
AT peiyeetiew evaluationofdropletdigitalpolymerasechainreactionddpcrfortheabsolutequantificationofiaspergillusispeciesinthehumanairway
AT sanjayhchotirmall evaluationofdropletdigitalpolymerasechainreactionddpcrfortheabsolutequantificationofiaspergillusispeciesinthehumanairway