报告噬菌体T6::Nluc的构建与诊断性能评估:一种快速检测大肠杆菌尿路感染的新方法OA

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.