Development of Duplex Droplet Digital PCR for Rapid Detection of Blood Stream Infections Caused by S. aureus, E. coli, S. pneumoniae and P. aeruginosa

Abstract Purpose: The aim of this study was to develop highly specific primer-probes pairs for the detection of species-specific genes, and establish a multiplex ddPCR assay for the detection of low copies of genomic DNA, of common bacteria causing bloodstream infections in our institution, i.e. Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, and Pseudomonas aerugnosa..Methods: Pubmed and GenBank databases were used to select well-characterized candidate genes for each microorganism. The newly designed primer-probe pairs were designed using AllelID 7.85 software. The designed primer-probe pairs were tested and validated using Droplet Digital PCR QX200 by using quantitative ATCC genomic DNA from S. aureus (ATCC® 29213DQ™), S. pneumoniae (ATCC® 700669DQ™), E. coli (ATCC® 25922DQ™), and P. aeruginosa (ATCC® 27853DQ™).Results: All primer-probe pairs demonstrated an optimal annealing temperature at 56°C. The ddPCR detected 162 copies for S. aureus and 152 copies S. pneumonia in singleplex reactions as compared to 152 copies and 132 copies in duplex reactions, respectively, and 324 copies for P. aeruginosa, 30 copies for E. coli in singleplex reactions as compared to 356 copies and 34 copies, respectively, in duplex reactions. The results of the limit of detection of the ddPCR assay was as low as 1 copy/reaction for S. aureus, E. coli, and P. aeruginosa and 2 copies/reaction for S. pneumonia. Conclusion: In this study, we have designed highly specific PP pairs for use in a ddPCR assay for the accurate and reliable detection and quantification of species-specific genes for four common blood culture pathogens in our institution.