Development of novel digital PCR assays for the rapid quantification of Gram-negative bacteria biomarkers using RUCS algorithm

• Published in: Methods (San Diego, Calif.) Vol. 232; pp. 72 - 80
• Main Authors: Bogožalec Košir, Alexandra, Alič, Špela, Tomič, Viktorija, Lužnik, Dane, Dreo, Tanja, Milavec, Mojca
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2024
• Subjects: Acinetobacter baumannii; Acinetobacter baumannii - genetics; Acinetobacter baumannii - isolation & purification; Algorithms; Biomarkers; Biomarkers - analysis; cross reaction; detection limit; diagnostic techniques; Digital PCR (dPCR); disease control; DNA; DNA, Bacterial - analysis; DNA, Bacterial - genetics; genome; Gram-negative bacteria; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - isolation & purification; Gram-Negative Bacterial Infections - diagnosis; Gram-Negative Bacterial Infections - microbiology; hospitals; Humans; Klebsiella pneumoniae; Klebsiella pneumoniae - genetics; Klebsiella pneumoniae - isolation & purification; matrix-assisted laser desorption-ionization mass spectrometry; Pathogen detection; Polymerase Chain Reaction - methods; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - isolation & purification; Reproducibility of Results; Respiratory infections; RUCS algorithm; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; uncertainty; Biomarkers; Pathogen detection; Digital PCR (dPCR); Gram-negative bacteria; RUCS algorithm; Respiratory infections
• ISSN: 1046-2023; 1095-9130; 1095-9130
• DOI: 10.1016/j.ymeth.2024.10.011

•RUCS-guided pipeline developed for genome informed assay design.•New biomarkers and dPCR methods for A. baumannii, K. pneumoniae, and P. aeruginosa.•Developed dPCR methods exhibit high sensitivity, accuracy, and efficiency in complex samples.•Validated against gold standard MALDI-TOF: 93.75% agreement in blind samples.•Potential for enhanced clinical diagnostics and infection control. Rapid and accurate identification of bacterial pathogens is crucial for effective treatment and infection control, particularly in hospital settings. Conventional methods like culture techniques and MALDI-TOF mass spectrometry are often time-consuming and less sensitive. This study addresses the need for faster and more precise diagnostic methods by developing novel digital PCR (dPCR) assays for the rapid quantification of biomarkers from three Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Utilizing publicly available genomes and the rapid identification of PCR primers for unique core sequences or RUCS algorithm, we designed highly specific dPCR assays. These assays were validated using synthetic DNA, bacterial genomic DNA, and DNA extracted from clinical samples. The developed dPCR methods demonstrated wide linearity, a low limit of detection (∼30 copies per reaction), and robust analytical performance with measurement uncertainty below 25 %. The assays showed high repeatability and intermediate precision, with no cross-reactivity observed. Comparison with MALDI-TOF mass spectrometry revealed substantial concordance, highlighting the methods’ suitability for clinical diagnostics. This study underscores the potential of dPCR for rapid and precise quantification of Gram-negative bacterial biomarkers. The developed methods offer significant improvements over existing techniques, providing faster, more accurate, and SI-traceable measurements. These advancements could enhance clinical diagnostics and infection control practices.