title: 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.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay.
authors.affiliation: Centre for Heart Lung Innovation, St. Paul's Hospital, Departments of Medicine and Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Sze
authors.affiliation: BioRad Laboratories, Mississauga, ON, Canada.
authors.forename: Meysam
authors.initials: M
authors.lastname: Abbasi
authors.affiliation: Centre for Heart Lung Innovation, St. Paul's Hospital, Departments of Medicine and Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: James C
authors.initials: JC
authors.lastname: Hogg
authors.affiliation: Centre for Heart Lung Innovation, St. Paul's Hospital, Departments of Medicine and Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Don D
authors.initials: DD
authors.lastname: Sin
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008168:Lung / Q000382:microbiology / Q000473:pathology; D064307:Microbiota / Q000235:genetics*; D016133:Polymerase Chain Reaction; D029424:Pulmonary Disease, Chronic Obstructive / Q000235:genetics / Q000382:microbiology* / Q000473:pathology; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S