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author:

Wu, H. (Wu, H..) [1] | He, J.-S. (He, J.-S..) [2] | Zhang, F. (Zhang, F..) [3] | Ping, J. (Ping, J..) [4] | Wu, J. (Wu, J..) [5]

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Scopus

Abstract:

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®) 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 °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. © 2019 Elsevier B.V.

Keyword:

CaMV35S promoter; CRISPR/Cas12a; LAMP reaction; Reaction vessel; Visual detection

Community:

  • [ 1 ] [Wu, H.]College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
  • [ 2 ] [He, J.-S.]College of Food Science and Technology, Yunnan Agricultural University, Kunming, 650201, China
  • [ 3 ] [Zhang, F.]College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, China
  • [ 4 ] [Ping, J.]College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
  • [ 5 ] [Wu, J.]College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China

Reprint 's Address:

  • [Wu, J.]College of Biosystems Engineering and Food Science, Zhejiang UniversityChina

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Source :

Analytica Chimica Acta

ISSN: 0003-2670

Year: 2020

Volume: 1096

Page: 130-137

6 . 5 5 8

JCR@2020

5 . 7 0 0

JCR@2023

ESI HC Threshold:160

JCR Journal Grade:1

CAS Journal Grade:2

Cited Count:

WoS CC Cited Count:

SCOPUS Cited Count: 64

ESI Highly Cited Papers on the List: 0 Unfold All

WanFang Cited Count:

Chinese Cited Count:

30 Days PV: 1

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