A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2

• Published in: PloS one Vol. 9; no. 10; p. e110351
• Main Authors: Sze, Marc A., Abbasi, Meysam, Hogg, James C., Sin, Don D.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.10.2014; Public Library of Science (PLoS)
• Subjects: Airway management; Analysis; Assaying; Background noise; Bacteria; Bacteria - genetics; Bacteria - isolation & purification; Bacteria - pathogenicity; Biology and Life Sciences; Chronic obstructive lung disease; Chronic obstructive pulmonary disease; Coefficient of variation; Control methods; Correlation analysis; Cystic fibrosis; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; E coli; Efficiency; Escherichia coli; Ethics; Gold; Histology; Humans; Laboratories; Lung - microbiology; Lung - pathology; Lung diseases; Medicine; Medicine and Health Sciences; Microbiomes; Microbiota - genetics; New technology; Noise reduction; Obstructive lung disease; Pathology; Polymerase Chain Reaction; Primers; Pulmonary Disease, Chronic Obstructive - genetics; Pulmonary Disease, Chronic Obstructive - microbiology; Pulmonary Disease, Chronic Obstructive - pathology; Research and Analysis Methods; RNA; RNA, Ribosomal, 16S - genetics; rRNA 16S; Surgery; Tissues; Canada
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0110351

Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR. Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue. There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05). Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay.