Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters
| Autor: | Felix von Stetten, Roland Zengerle, Mohammed A. Bakheit, Nadine Borst, Silvina Stinco, Lisa Becherer, Sieghard Frischmann |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Chemistry Loop-mediated isothermal amplification 02 engineering and technology 021001 nanoscience & nanotechnology Dna amplification Fluorescence Displacement (vector) Analytical Chemistry Fluorescence ratio 03 medical and health sciences Fluorescence intensity 030104 developmental biology Molecular beacon Multiplex 0210 nano-technology Biological system |
| Zdroj: | Analytical Chemistry. 90:4741-4748 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.7b05371 |
| Popis: | A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels. |
| Databáze: | OpenAIRE |
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