Journal
ANALYTICA CHIMICA ACTA
Volume 1096, Issue -, Pages 130-137Publisher
ELSEVIER
DOI: 10.1016/j.aca.2019.10.042
Keywords
CRISPR/Cas12a; CaMV35S promoter; LAMP reaction; Visual detection; Reaction vessel
Categories
Funding
- National Natural Science Foundation of China [31571918]
- Key Research & Development Programs in Yunnan Province [2018BC005]
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An ultrafast and convenient method for visually detecting CaMV35S promoter amplicon (amplified products) was established by using CRISPR/Cas12a system coupled with a designed reaction vessel. Genetically modified (GM) soybean (Roundup Ready (R)) powders containing CaMV35S promoter were employed as detection targets, which were amplified by loop-mediated isothermal amplification (LAMP). The CRISPR/Cas12a system directly mixed with amplified products at 37 degrees C for 5 min and detection results could be clearly identified by the naked eye under UV light (254 nm). A designed reaction vessel was employed to make operation easier and could effectively prevent contamination at the source. The CRISPR/Cas12a detection system was optimized in our study and the concentration of magnesium ions was proved to be important for the work of CRISPR/Cas12a system. The optimized concentration range of magnesium ions was between 10 mM and 12 mM. Besides, the activated Bst DNA polymerase also had little effects on CRISPR/Cas12a system. The developed method could significantly distinguish the specific and non-specific amplification. And as low as 0.05% transgenic contents in soybean powders could be detected. It would have the potential to be complementary to instrument-based ultrahigh sensitive method and provide a new solution for on-site rapid detection. (C) 2019 Elsevier B.V. All rights reserved.
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