An enzyme free fluorescence resonance transfer strategy based on hybrid chain reaction and triplex DNA for Vibrio parahaemolyticus

Xiao-Hui Tan,Yu-Bin Li,Yan Liao,Hua-Zhong Liu

doi:10.1038/s41598-020-77913-2

Xiao-Hui Tan, Yu-Bin Li + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-77913-2

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Journal: Scientific Reports	Publication Date: Nov 26, 2020
Citations: 7	License type: open-access

Affiliation: Guangdong Ocean University

Abstract

In this work, an enzyme-free fluorescence resonance energy transfer (FRET) strategy was established for rapid and specific detection of the DNA sequence from Vibrio parahaemolyticus (VP) using hybridization chain reaction (HCR) amplification and triplex DNA. The triplex forming oligonucleotide (TFO) was labelled with carboxyfluorescein (FAM) as fluorescence donor, and hairpin sequence H1 was labelled by tetramethylrhodamine (TAMRA) as fluorescence receptor. In the present target VP DNA, the hairpin structure of molecular beacon (MB) was opened, the free end was released and hybridized with H1-TAMRA, and the HCR reaction was triggered by the alternate supplementation of H1-TAMRA and H2 to produce the notch double helix analogue. After the addition of TFO-FAM, a triplex structure was formed between HCR products (H1-TAMRA/H2) and TFO-FAM. A close contact between the donor and the receptor resulted in FRET. Under the optimal conditions, the fluorescence quenching value was inversely proportional to the concentration of target VP DNA in the range of 0.1–50 nmol L−1, and the detection limit was 35 pmol L−1.

Highlights

Vibrio parahaemolyticus (VP) is widely distributed in seawater and seafood such as shrimp, crab, shellfish, etc., which can cause gastroenteritis, wound infection and septicemia
With the purpose of rapid detection of VP, we develop an assay for the specific detection of VP DNA sequence
With the addition of target VP DNA, the hairpin structure of molecular beacon (MB) is opened and the free end is released and hybridized with Hairpin probe 1 (H1)-TAMRA, the hybridization chain reaction (HCR) reaction is triggered by the alternate supplementation of H1-TAMRA and Hairpin probe 2 (H2) to produce double-stranded DNA (dsDNA)

Summary

Introduction

Vibrio parahaemolyticus (VP) is widely distributed in seawater and seafood such as shrimp, crab, shellfish, etc., which can cause gastroenteritis, wound infection and septicemia. The HCR products can be recognized by TFO-FAM and form triplex DNA. The TAMRA and FAM are brought closer together by the formation of triplex DNA, resulting in a FRET process and a dramatic decrease in the fluorescence intensity at 520 nm.

Results

Conclusion