To develop a visual, sensitive, and specific method for the determination of Escherichia coli O157:H7 in food, the rfbE gene, specific to the O157:H7 serotype, was selected as the target sequence.A circular probe was designed to recognize target sequences.After DNA binding, the circular probe was circularized by ligation and then hybridize with capture probes.Capture probes act as primers to initiate rolling circle amplification (RCA) catalyzed by Phi29 DNA polymerase, generating a long single-stranded DNA (ssDNA).SYBR Green Ⅱ binding to the ssDNA resulted in significantly enhanced fluorescence, with signal intensity proportional to the ssDNA concentration.This enabled real-time quantitative monitoring in laboratory settings, while on-site qualitative detection was achieved through visual fluorescence under UV light.The method demonstrated a wide linear detection range (5×10-3 to 5×10-1 ng/μL) for genomic DNA.The detection limits were 7.5×10-3 ng/μL (visual mode) and 5.048×10-3 ng/μL (real-time fluorescence mode).Excellent specificity was confirmed by the absence of cross-reactivity with non-O157:H7 E.coli strains or other bacterial species.The potential application of the method was verified by analyzing spiked food samples.The developed dual-mode detection system offers significant advantages for rapid, sensitive, and specific detection in food safety monitoring.
ZHANG Jingxin
,
TANG Jianing
,
HAN Bingyan
,
WANG Yijia
,
ZHANG Siqi
,
CHI Linye
,
WANG Jie
,
HAN Aiyun
,
DENG Rui
,
LI Yanan
,
CHEN Yongmin
. A real-time quantitative fluorescence/UVA irradiation visualized dual-modal signal output biosensor based on rolling circle amplification for determination of Escherichia coli O157:H7[J]. Food and Fermentation Industries, 2026
, 52(4)
: 368
-375
.
DOI: 10.13995/j.cnki.11-1802/ts.043208
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