陆军军医大学学报2026，Vol.48Issue(2)：147-157,11.DOI:10.16016/j.2097-0927.202510022
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
Construction of reporter phage T6::Nluc and evaluation of its diagnostic performance:A novel method for rapid detection of Escherichia coli urinary tract infections
摘要
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.关键词
报告噬菌体/大肠杆菌/尿路感染/CRISPR/Cas9/病原菌检测Key words
reporter phage/Escherichia coli/urinary tract infections/CRISPR/Cas9/pathogen detection分类
生物科学引用本文复制引用
郝治云,赵强,李民伟,李绵洋,王驰,王成彬..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[J].陆军军医大学学报,2026,48(2):147-157,11.基金项目
国家自然科学基金项目面上项目(82172364),北京市自然科学基金项目(L244076) Supported by the General Program of National Natural Science Foundation of China(82172364)and the Natural Science Foundation of Beijing(L244076). （82172364）
