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. 2025 Feb;104(2):104745.
doi: 10.1016/j.psj.2024.104745. Epub 2024 Dec 28.

Rapid detection of Pan-Avian Influenza Virus and H5, H7, H9 subtypes of Avian Influenza Virus using CRISPR/Cas13a and lateral flow assay

Yujia Yang  1 , Zhiyi Yang  1 , Xinkui Zhang  1 , Beibei Niu  1 , Qiuhong Huang  1 , Yan Li  2 , Huifang Yin  2 , Xianpeng Zhang  3 , Ming Liao  1 , Weixin Jia  4
Affiliations

Rapid detection of Pan-Avian Influenza Virus and H5, H7, H9 subtypes of Avian Influenza Virus using CRISPR/Cas13a and lateral flow assay

Yujia Yang et al. Poult Sci. 2025 Feb.

Abstract

Avian Influenza Virus (AIV) has been prevalent worldwide in recent years, resulting in substantial economic losses in the poultry industry. More importantly, AIV is capable of cross-species transmission among mammals, posing a dormant yet considerable threat to human health and safety. In this study, two rapid detection methods for AIV based on the CRISPR-Cas13a were developed. These methods can identify AIV through the M gene and differentiate the H5, H7, and H9 subtypes via the HA gene. The first method utilizes RT-RAA isothermal amplification of the target sequence in combination with the "collateral effect" of the Cas13a protein. The results are measured using a real-time quantitative PCR instrument, with a Limit of Detection (LOD) as low as 1 copy/μL. The second method combines RT-RAA with Cas13a and a lateral flow assay, allowing results to be visually observed with the naked eye, with a LOD of 10 copies/μL. Both methods demonstrated specificity and sensitivity comparable to or exceeding that of qRT-PCR, suggesting strong potential for clinical application.

Keywords: Avian influenza virus; CRISPR/Cas13a; Lateral flow assay; RT-RAA; Rapid detection.

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Conflict of interest statement

Declaration of competing interest The authors declare no conflicts of interest.

Figures

Fig 1
Fig. 1
Schematic of RT-RAA-CRISPR-Taqman and RT-RAA-CRISPR-LFD
Fig 2
Fig. 2
Analysis of crRNA consistency,a-d represents crRNA targeting H5, H7, H9 and M genes, respectively.
Fig 3
Fig. 3
Schematic diagram of RT-RAA-CRISPR-LFD test strip result interpretation
Fig 4
Fig. 4
RT-RAA amplification results. M represents Marker, where 1 in a,b,c represents the positive sample and 2 represents the NC. In the d, 1,2,3,4,5,6 represent the positive sample, and 7 represents the NC.
Fig 5
Fig. 5
RT-RAA CRISPR-TaqMan-M condition optimization results.
Fig 6
Fig. 6
RT-RAA CRISPR-TaqMan-H5 condition optimization results.
Fig 7
Fig. 7
RT-RAA CRISPR-TaqMan-H7 condition optimization results.
Fig 8
Fig. 8
RT-RAA CRISPR-TaqMan-H9 condition optimization results
Fig 9
Fig. 9
The Specificity of RT-RAA-CRISPR-Taqman detection melthod. The a,b,c and d represent the results of M, H5, H7, and H9, respectively.
Fig 10
Fig. 10
The Sensitivity of RT-RAA-CRISPR-Taqman detection melthod. The a,b,c and d represent the results of M, H5, H7, and H9, respectively.
Fig 11
Fig. 11
The Specificity of RT-RAA-CRISPR-LFD detection melthod. The a,b,c and d represent the results of M, H5, H7, and H9, respectively.
Fig 12
Fig. 12
The Sensitivity of RT-RAA-CRISPR-LFD detection melthod. The a,b,c and d represent the results of M, H5, H7, and H9, respectively.
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