Construction of reporter phage T6::Nluc and evaluation of its diagnostic performance: A novel method for rapid detection of Escherichia coli urinary tract infections

Abstract Objective To construct an Escherichia coli (E. coli)-specific reporter phage, T6::Nluc, and evaluate its performance in the rapid diagnosis of E. coli-induced urinary tract infections. Methods T6::Nluc was constructed by the CRISPR/Cas9 system combined with homologous recombination technology. Then biological characteristics of T6 and T6::Nluc were systematically compared in terms of optimal multiplicity of infection (MOI), one-step growth curves, and lysis efficiency. Time-luminescence curves were plotted to determine the limit of detection (LOD) and the impact of urine matrix. Luminescence quantification was performed to detect the specificity of the phage after being co-cultured with non-E.coli pathogens. In addition, 104 strains of E. coli isolated from clinical urine samples were collected to analyze the detection coverage of T6::Nluc. Finally, 437 clinical urine samples were obtained for clinical validation. Results The successful construction of reporter phage T6::Nluc was validated by Sanger sequencing. Its one-step growth curve and optimal MOI differed from those of T6, and the lysis efficiency remained comparable. T6::Nluc exhibited outstanding diagnostic performance, enabling the detection of ≥10⁴ CFU/mL E. coli within 40 min, with complete resistance to interference from urine matrix and excellent pathogen-specific recognition. Additionally, T6::Nluc successfully detected 51 out of 104 clinical E. coli isolates (49.04%). And the results of the detection in clinical urine samples indicated that the phage had a sensitivity of 48.84% and a specificity of 100.00 in the detection. Conclusion Reporter phage T6::Nluc is successfully constructed, and can sensitively and specifically detect E. coli causing urinary tract infections, significantly reducing detection time and enhancing efficiency and accuracy.