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

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

Zongshu Zhang #  1 , Chunguang Wang #  1 , Xi Chen  1 , Zichuang Zhang  2 , Guoqiang Shi  3 , Xianghe Zhai  1 , Tie Zhang  1
Affiliations

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

Zongshu Zhang et al. Front Vet Sci. .

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.

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Conflict of interest statement

GS was employed by Hebei Sanshi Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic of primers and crRNA design. (A) The alignment results of NP sequences from different subtypes (H5, H7, H9) of AIV; (B) Schematic of primers and crRNA design.
Figure 2
Figure 2
Specificity test results of RAA-CRISPR-Cas13a-LFD method for AIV detection. NC, negative control. Only AIV-H5, AIV-H7, and AIV-H9 showed T and C lines, while other viruses and negative control only showed C line.
Figure 3
Figure 3
Sensitivity test results of the three methods for AIV detection. (A–C) Were the results of AIV detection by RAA-CRISPR-Cas13a-LFD method, PCR-agarose electrophoresis method, and qPCR method, respectively. NC, negative control. In (A), the lowest detection limit of RAA-CRISPR-Cas13a-LFD method was 100 copies/μL. In (B), the lowest detection limit of PCR-agarose electrophoresis method was 103 copies/μL. In (C), the lowest detection limit of qPCR was 101 copies/μL.
Figure 4
Figure 4
Reproducibility test results of RAA-CRISPR-Cas13a-LFD method for AIV detection. (A–C) Were the results of three replicate tests at template concentrations of 105, 103, and 101 copies/μL, respectively. NC, negative control. The T and C lines appeared in all three replicate groups, and only the C line appeared in the negative control.
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