Immuno-HCR based on contact quenching and fluorescence resonance energy transfer for sensitive and low background detection of Escherichia coli O157:H7

Escherichia coli O157:H7 makes a major threat to human health. Aiming to detect Escherichia coli O157:H7 sensitively, hybridization chain reaction signal amplified immunoassay (immuno-HCR) based on contact quenching (CQ) and fluorescence resonance energy transfer (FRET) was developed. The background of the new designed HCR hairpins (CQ-FRET hairpins) was reduced by contact-quenching fluorescein (FAM) and breaking FRET from donor (FAM) to acceptor (Cy5). The F/F-0 ratio of CQ-FRET hairpins (37.02) was obviously higher than that of two other common HCR fluorescent hairpins (CQ hairpins, 21.45; FRET hairpins, 4.61). The limit of detection of the assay was 3.5 x 10(1) CFU/mL and obviously lower than that of CQ hairpins based immuno-HCR (3.28 x 10(3) CFU/mL) and FRET hairpins based immuno-HCR (6.49 x 10(4) CFU/mL). The proposed low fluorescent background immuno-HCR with high sensitivity which was verified in contaminated milk samples could be potentially used in the detection of various pathogens.

大肠杆菌O157:H7对人类健康构成重大威胁。为了灵敏地检测大肠杆菌O157:H7，基于接触猝灭（CQ）和荧光共振能量转移（FRET）开发了杂交链式反应信号放大免疫测定法（免疫 - HCR）。通过接触猝灭荧光素（FAM）以及阻断从供体（FAM）到受体（Cy5）的FRET，降低了新设计的HCR发夹（CQ - FRET发夹）的背景。CQ - FRET发夹的F/F₀比值（37.02）明显高于其他两种常见的HCR荧光发夹（CQ发夹，21.45；FRET发夹，4.61）。该测定法的检测限为3.5×10¹ CFU/mL，明显低于基于CQ发夹的免疫 - HCR（3.28×10³ CFU/mL）和基于FRET发夹的免疫 - HCR（6.49×10⁴ CFU/mL）。所提出的这种在污染牛奶样本中得到验证的低荧光背景且具有高灵敏度的免疫 - HCR，有可能用于各种病原体的检测。