Dual-Gene Isothermal Amplification Coupled with Lateral Flow Strip for On-Site Accurate Detection of E. coli O157:H7 in Food Samples

放大器 大肠杆菌 基因组DNA 化学 重组酶聚合酶扩增 基因 聚合酶链反应 受污染的食物 DNA 核酸 分子生物学 计算生物学 生物化学 微生物学 生物
作者
Ting Zheng,Xianming Li,Yani Xie,Bin Yang,Peng Wu
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:95 (14): 6053-6060 被引量:12
标识
DOI:10.1021/acs.analchem.3c00141
摘要

On-site field detection of E. coli O157:H7 in food samples is of utmost importance, since it causes a series of foodborne diseases due to infections-associated ready-to-eat foods. Due to the instrument-free nature, recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) is well-suited for such goal. However, the high genomic similarity of different E. coli serotypes adds difficulty to accurate differentiation of E. coli O157:H7 from others. Dual-gene analysis could significantly improve the serotype selectivity, but will further aggravate the RPA artifacts. To address such issue, here we proposed a protocol of dual-gene RPA-LFA, in which the target amplicons were selectively recognized by peptide nucleic acid (PNA) and T7 exonuclease (TeaPNA), thus eliminating false-positives in LFA readout. Adapting rfbEO157 and fliCH7 genes as the targets, dual-gene RPA-TeaPNA-LFA was demonstrated to be selective for E. coli O157:H7 over other E. coli serotypes and common foodborne bacteria. The minimum detection concentration was 10 copies/μL for the genomic DNA (∼300 cfu/mL E. coli O157:H7), and 0.24 cfu/mL E. coli O157:H7 in food samples after 5 h bacterial preculture. For lettuce samples contaminated with E. coli O157:H7 (single-blind), the sensitivity and specificity of the proposed method were 85% and 100%, respectively. Using DNA releaser for fast genomic DNA extraction, the assay time could be reduced to ∼1 h, which is appealing for on-site food monitoring.
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