Abstract
BACKGROUND: Bloodstream infection (BSI) is associated with high morbidity and mortality worldwide. Currently, BSI diagnosis relies on a time-consuming blood culture method, which usually takes 2 or more days to identify the causative pathogens. Cell-free DNA (cfDNA) refers to those small nucleic acid fragments residing in plasma and other body fluids, which have been used to detect cancer, organ transplantation injury, and pathogenic infections. A new multiplexed fluorescent quantitative PCR kit aiming at plasma microbial cfDNA was developed in this study. The kit contains multiple panels, and each panel covers multiple pathogens, including E. coli, K. pneumoniae, A. baumannii, H. influenzae, P. aeruginosa, E. faecalis, E. faecium, S. aureus, and S. epidermidis. METHODS: PCR primers and probes were designed based on effective bacterial sequence segments, which were obtained from the analysis of next-generation sequencing results of plasma samples of patients with bloodstream infections. Bioinformatics analyses and experimental evidence were used to test the conservativeness and specificity of the primers and probes. The lower detection limit of the kit was determined under optimal reaction conditions. Clinical samples were used to test the accuracy of the kit's detection. RESULTS: The lower detection limit of the kit has reached ≤ 12 copies per reaction. Clinical samples testing results showed a 90.48% consistency between the kit and blood culture. CONCLUSION: The kit provides a rapid, accurate, and reliable method for diagnosing bloodstream infections. This can quickly provide an etiological basis for clinical diagnosis and further treatment, potentially improving patient outcomes.