Multiple cross displacement amplification coupled with a lateral flow biosensor for ultra-rapid and highly sensitive detection of Escherichia coli in neonatal sepsis

Escherichia coli ( E. coli ) is a predominant pathogen causing neonatal sepsis, and rapid pathogen identification is critical for timely antimicrobial therapy. However, conventional diagnostic methods are often limited by prolonged turnaround time, or insufficient sensitivity. In this study, we developed a multiple cross-displacement amplification assay coupled with a lateral flow biosensor (MCDA-LFB) targeting the uidA gene for rapid visual detection of E. coli . Using a set of 10 specific primers, the reaction conditions were optimized at 63°C for 40 min. The analytical limit of detection was 50 fg genomic DNA per reaction, and no cross-reactivity was observed among the tested non– E . coli strains. Clinical performance was evaluated using 75 residual sterile body fluid specimens, including 58 blood and 17 cerebrospinal fluid samples, collected from neonates who were clinically diagnosed with sepsis or bacterial meningitis, and in whom E. coli infection was considered highly likely on the basis of the overall clinical assessment. Results were compared with quantitative PCR (qPCR) and conventional culture. In this cohort, the MCDA-LFB assay showed complete agreement with qPCR ( κ = 1.0) and detected 56.0% (42/75) positive samples, compared with 14.7% (11/75) by culture ( P < 0.001). The entire workflow, including nucleic acid extraction, amplification, and visual readout, was completed within approximately 1 h. These findings suggest that the E. coli -MCDA-LFB assay provides a rapid, sensitive, and cost-effective approach for E. coli detection and may be useful for early diagnosis in clinical neonatal sepsis, particularly in point-of-care and resource-limited settings. IMPORTANCE Neonatal sepsis requires rapid and accurate pathogen identification to support timely antimicrobial therapy, yet conventional blood culture is often slow and has limited sensitivity, particularly in neonates with small sample volume or prior antibiotic exposure. In this study, we developed a visual multiple cross displacement amplification–lateral flow biosensor (MCDA-LFB) assay for rapid detection of Escherichia coli , a leading cause of neonatal sepsis. The assay enables visual readout within approximately 1 h, shows femtogram-level analytical sensitivity, and demonstrates complete agreement with quantitative PCR (qPCR) in our clinical validation cohort. Owing to its operational simplicity and minimal equipment requirements, this method may serve as a useful approach for rapid detection of neonatal E. coli infection, particularly in resource-limited clinical microbiology settings.