Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Zongshu Zhang; Chunguang Wang; Xi Chen; Zichuang Zhang; Guoqiang Shi; Xianghe Zhai; Tie Zhang

doi:10.3389/fvets.2023.1272612

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses

Front Vet Sci. 2024 Jan 8:10:1272612. doi: 10.3389/fvets.2023.1272612. eCollection 2023.

Authors

Zongshu Zhang^#¹, Chunguang Wang^#¹, Xi Chen¹, Zichuang Zhang², Guoqiang Shi³, Xianghe Zhai¹, Tie Zhang¹

Affiliations

¹ College of Veterinary Medicine, Hebei Agricultural University, Baoding, China.
² Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, China.
³ Hebei Sanshi Biotechnology Co., Ltd., Shijiazhuang, China.

^# Contributed equally.

Abstract

To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 10⁰ copies/μL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

Keywords: CRISPR associated proteins; Clustered Regularly Interspaced Short Palindromic Repeats; avian influenza virus; lateral flow dipstick; nucleic acid detection; recombinase-aided amplification.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by S&T Program of Hebei (No. 22326608D), Hebei Province, China, and the High-level Science and Technology Innovation and Entrepreneurship Talent Project of Shijiazhuang (No. 05202001), Shijiazhuang, China. The funding bodies played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript.