研究目的
The aim of this study is to design fluorometric assay specifically for the detection of fliC gene. The fliC gene that encodes the E. coli structural flagella antigen (H7) provides genotypic identification of the H7 serotype most commonly associated with disease.
研究成果
A sensitive and practical fluorometric assay has been successfully designed for the detection of E. coli O157:H7 based on fluorescence quenching. The method indicates notable features, including sensitivity, selectivity, and quantitative capability, proving its potential for use in food safety to identify pathogenic bacteria.
研究不足
The system requires a long tracking time of about 4 h and is not stable for analysis of samples that have long target sequence due to steric hindrance. It is not suitable for point of care application as the sample needs to be brought to the laboratory and special dedicated instrument is required.
1：Experimental Design and Method Selection:
A fluorometric assay was designed by combining the unique properties of GQDs with AuNPs to determine the fliC gene of E. coli O157:H7. The assay involved the conjugation of reporter oligos on GQDs and quencher oligos on AuNPs, followed by co-hybridization with target DNA to trigger fluorescence quenching.
2：The assay involved the conjugation of reporter oligos on GQDs and quencher oligos on AuNPs, followed by co-hybridization with target DNA to trigger fluorescence quenching.
Sample Selection and Data Sources:
2. Sample Selection and Data Sources: The study used DNA oligos designed for the fliC gene of E. coli O157:H7, synthesized and purified by Integrated DNA Technologies (IDT) Inc. Food samples (chicken, meat, and cheese) spiked with E. coli O157:H7 were used for real sample analysis.
3：List of Experimental Equipment and Materials:
GQDs nanoparticles, gold nanoparticles (AuNPs), 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were used. A Synergy H1 Hybrid Multi-Mode microplate reader was used for fluorescence measurement.
4：Experimental Procedures and Operational Workflow:
The process included preparation of thiolated AuNPs and modified amine-GQDs, fluorescence quenching experiments, and application in real samples. The fluorescence signal was determined with excitation/emission peaks at 400 nm/530 nm.
5：Data Analysis Methods:
The fluorescence quenching efficiency was calculated, and the limit of detection (LOD) was determined based on the calibration plot.
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