Rapid detection of H5 subtype avian influenza virus using CRISPR Cas13a based-lateral flow dipstick

Yang Li; Jiajing Shang; Juan Luo; Fuyou Zhang; Ge Meng; Yingjie Feng; Wenming Jiang; Xiaohui Yu; Chunran Deng; Guanhui Liu; Hualei Liu

doi:10.3389/fmicb.2023.1283210

Rapid detection of H5 subtype avian influenza virus using CRISPR Cas13a based-lateral flow dipstick

Front Microbiol. 2023 Nov 29:14:1283210. doi: 10.3389/fmicb.2023.1283210. eCollection 2023.

Authors

Yang Li^#¹, Jiajing Shang^#^{1

2}, Juan Luo^{1

2}, Fuyou Zhang¹, Ge Meng^{1

2}, Yingjie Feng^{1

2}, Wenming Jiang¹, Xiaohui Yu¹, Chunran Deng^{1

2}, Guanhui Liu², Hualei Liu¹

Affiliations

¹ China Animal Health and Epidemiology Center, Qingdao, China.
² School of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, China.

^# Contributed equally.

Abstract

Due to its high mortality rate, highly pathogenic avian influenza (HPAI), a notifiable animal illness designated by the World Organisation for Animal Health (WOAH), has caused enormous financial losses to the poultry sector. The H5 subtype of avian influenza virus (H5-AIV) is regarded as the most common highly pathogenic avian influenza virus (HPAIV) that threatens public health and safety. Virus isolation and reverse transcription quantitative PCR (RT-qPCR) are usually used to detect H5-AIV and are important for the timely diagnosis and control of H5-AIV. However, these methods are time-consuming and require a significant amount of effort. In this study, we established a recombinase-aided amplification (RAA) combined with CRISPR-Cas13a and lateral flow dipstick (LFD) assay for the detection of H5-AIV. The results showed that the process can be completed within 40 min at 37°C. The method had a detection limit of 0.1 copy/μL, which was comparable to the RT-qPCR. There was no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, H10-AIV, IBV, NDV, RVA and DAstV. The kappa value of RT-RAA-Cas13a-LFD and RT-qPCR in 380 clinical samples was 0.89 (κ>0.75). In conclusion, we established a convenient, efficient and accurate method to detect H5-AIV, and the results can be visualized and interpreted using LFD, which can be adapted to the needs of grassroots laboratories and field-deployable assays. This approach provides a new perspective for clinical H5-AIV diagnosis and has great potential for application in clinical quarantine of the poultry farming.

Keywords: CRISPR Cas13a; H5-AIV; RAA; lateral flow dipstick; rapid diagnosis.

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 the Shandong Provincial Key Research and Development Program (2022CXGC010606).