Evaluation of Droplet Digital Polymerase Chain Reaction (ddPCR) for the Absolute Quantification of Aspergillus species in the Human Airway

• Published in: International journal of molecular sciences 2020-04, Vol.21 (9), p.3043
• Main Authors: Poh, Tuang Yeow, Ali, Nur A'tikah Binte Mohamed, Chan, Louisa L Y, Tiew, Pei Yee, Chotirmall, Sanjay H
• Format: Article
• Language: English
• Subjects: Abundance; Aged; Aspergillus; Aspergillus - genetics; Aspergillus - isolation & purification; Bacterial Load; Bronchiectasis; Bronchiectasis - microbiology; Case-Control Studies; Chronic obstructive pulmonary disease; Cystic fibrosis; Deoxyribonucleic acid; DNA; DNA polymerase; DNA, Fungal - genetics; Droplets; Early Diagnosis; Evaluation; Female; Fungi; Genes; Humans; Limit of Detection; Lung diseases; Male; Middle Aged; Obstructive lung disease; Plasmids; Polymerase chain reaction; Polymerase Chain Reaction - methods; Prospective Studies; Pulmonary Aspergillosis - diagnosis; Pulmonary Disease, Chronic Obstructive - microbiology; Real-Time Polymerase Chain Reaction; Respiratory diseases; Respiratory tract; Sensitivity and Specificity; Sputum; Sputum - microbiology
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21093043

Prior studies illustrate the presence and clinical importance of detecting 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 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 when compared to standard qPCR. The molecular approaches of qPCR and ddPCR were applied to DNA fungal extracts in = 20 sputum specimens obtained from non-diseased ( = 4), chronic obstructive pulmonary disease (COPD; = 8) and non-cystic fibrosis bronchiectasis ( = 8) patients where status was known. DNA was extracted and qPCR and ddPCR performed on all specimens with appropriate controls and head-to-head comparisons performed. Standard qPCR and ddPCR were both able to detect, even at low abundance, species ( and ) from specimens known to contain the respective fungi. Importantly, however, ddPCR was superior for the detection of particularly when present at very low abundance and demonstrates greater resistance to PCR inhibition compared to qPCR. ddPCR has greater sensitivity for detection from respiratory specimens, and is more resistant to PCR inhibition, important attributes considering the importance of species in chronic respiratory disease states such as bronchiectasis.