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Volume 13
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10.3390/foods13233816
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Article

Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis

by
Sisi Huang
1,
Shihui Wang
1,
Tianlong Wang
1,
Hongwei Song
1,
Yan Guo
1,
Xiong Xiong
1,* and
Libin Wang
2
1
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China
2
College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Foods 2024, 13(23), 3816; https://doi.org/10.3390/foods13233816
Submission received: 27 October 2024 / Revised: 19 November 2024 / Accepted: 25 November 2024 / Published: 26 November 2024
(This article belongs to the Special Issue Food Safety Detection Analysis and Sensors)
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Abstract

The self-quenching fluorogenic probe facilitates precise identification of LAMP (loop-mediated isothermal amplification) amplicons, unaffected by non-specific products resulting from primer dimers. However, low quenching efficiency by surrounding nucleobases leads to high background signal, posing significant challenges for visual inspection with the naked eye. The present study aims to identify an oligonucleotide sequence that is complementary to the self-quenching fluorogenic probe, and to employ the fluorescence super-quenching mechanism of double-stranded DNA to establish a visualization system for the LAMP assay. The results indicated that the incorporation of a sequence fully complementary to the probe could significantly reduce the system’s background fluorescence (p < 0.05). When the melting temperature exceeds room temperature, truncating the complementary sequence from the 3′ end does not compromise the probe’s quenching efficiency. The LAMP visualization system, using a 10–13-base complementary sequence of the loop primer-based probe, could effectively minimize background fluorescence and yield straightforward visual results post-reaction. Applied to rainbow trout and Atlantic salmon detection, the system detected 1 pg DNA in a closed-tube format. In conclusion, a suitable complementary sequence can reduce the background fluorescence of the self-quenching fluorogenic probe. Employing this sequence alongside the self-quenching fluorogenic probe to develop a low-background fluorescence LAMP system demonstrates great potential for successful visual detection and holds considerable promotional merit.
Keywords: loop-mediated isothermal amplification; self-quenching fluorogenic probe; visual inspection; sequence-specific detection; on-site detection loop-mediated isothermal amplification; self-quenching fluorogenic probe; visual inspection; sequence-specific detection; on-site detection

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MDPI and ACS Style

Huang, S.; Wang, S.; Wang, T.; Song, H.; Guo, Y.; Xiong, X.; Wang, L. Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis. Foods 2024, 13, 3816. https://doi.org/10.3390/foods13233816

AMA Style

Huang S, Wang S, Wang T, Song H, Guo Y, Xiong X, Wang L. Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis. Foods. 2024; 13(23):3816. https://doi.org/10.3390/foods13233816

Chicago/Turabian Style

Huang, Sisi, Shihui Wang, Tianlong Wang, Hongwei Song, Yan Guo, Xiong Xiong, and Libin Wang. 2024. "Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis" Foods 13, no. 23: 3816. https://doi.org/10.3390/foods13233816

APA Style

Huang, S., Wang, S., Wang, T., Song, H., Guo, Y., Xiong, X., & Wang, L. (2024). Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis. Foods, 13(23), 3816. https://doi.org/10.3390/foods13233816

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