Quantification of bacterial DNA in blood using droplet digital PCR: a pilot study

Abstract

AimTo use genus/species-specific genes droplet digital PCR (ddPCR) assays to detect/quantify bacterial DNA from Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus spp in blood samples. Methods and ResultsBacterial DNA from clinical strains (4<n<12) was extracted, quantified and diluted (10-0.0001ng/L) and ddPCR assays were performed in triplicate. These ddPCR assays showed low replication variability, low detection limit (1-0.1pg/L) and high genus/species specificity. ddPCR assays were also used to quantify bacterial DNA obtained from spiked blood (1x104-1CFU/mL) of each bacterial genus/species. Comparison between ddPCR assays and bacterial culture was performed by Pearson correlation. There was an almost perfect correlation (r[≥]0.997, p[≤]0.001) between the number of CFU/mL from bacterial culture and the number of gene copies/mL detected by ddPCR. The time from sample preparation to results was determined to be 3.5-4h. ConclusionsThe results demonstrated the quantification capacity and specificity of the ddPCR assays to detect/quantify four of the most important bloodstream infection (BSI) bacterial pathogens directly from blood. Significance and ImpactThis pilot study results reinforce the potential of ddPCR for the diagnosis and/or severity stratification of BSI. Applied to patients blood samples it can improve diagnosis and diminish sample-to-results time, improving patient care.