Visual and label-free ASFV and PCV2 detection by CRISPR-Cas12a combined with G-quadruplex

Ying Wang; Rong Li; Yang Zhang; Weida Zhang; Sishun Hu; Zili Li

doi:10.3389/fvets.2022.1036744

Visual and label-free ASFV and PCV2 detection by CRISPR-Cas12a combined with G-quadruplex

Front Vet Sci. 2022 Nov 29:9:1036744. doi: 10.3389/fvets.2022.1036744. eCollection 2022.

Authors

Ying Wang^{1

2}, Rong Li¹, Yang Zhang¹, Weida Zhang¹, Sishun Hu^{1

3

4}, Zili Li^{1

3

4}

Affiliations

¹ State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
² College of Veterinary Medicine, Shanxi Agricultural University, Jinzhong, China.
³ Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan, China.
⁴ Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People's Republic of China, Wuhan, China.

Abstract

African swine fever (ASF) and postweaning multisystemic wasting syndrome (PMWS) are acute infectious diseases caused by the African swine fever virus (ASFV) and porcine circovirus type 2 (PCV2). At present, there are no effective vaccines for the prevention of ASFV. PMWS, which is harmful to the domestic and even the world pig industry, is difficult to cure and has a high mortality. So, developing simple, inexpensive, and accurate analytical methods to detect and effectively diagnose ASFV and PCV2 can be conducive to avoid ASFV and PCV2 infection. CRISPR has become a potentially rapid diagnostic tool due to recent discoveries of the trans-cleavage properties of CRISPR type V effectors. Herein, we report the visual detection based on CRISPR-Cas12a (cpf1), which is more convenient than fluorescence detection. Through in vitro cleavage target DNA activation, Cas12a can trans-cleavage ssDNA G-quadruplex. TMB/H₂O₂ and Hemin cannot be catalyzed by cleavaged G-DNA to produce green color products. This protocol is useful for the detection of ASFV and PCV2 with high sensitivity. This method can enable the development of visual and label-free ASFV and PCV2 detection and can be carried out in the field without relying on instruments or power. This method can complete nucleic acid detection at 37 °C without using other instruments or energy. Our research has expanded the application of Cas12a and laid the foundation for the field's rapid detection of viral nucleic acid in future.

Keywords: ASFV; Cas12a; G-quadruplex; PCV2; detection.