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

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.

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a

“…Li et al created two H5 subtype AIVs assays using realtime fluorescence reverse transcription recombinase-aided amplification (RF-RT-RAA) and RT-RAA coupled with lateral flow dipstick (RT-RAA-LFD), the lowest detectable limits were 1 copies/µL [41]. And they also developed a H5 subtype AIVs assay based on CRISPR/Cas13a, RT-RAA, and LFD with a sensitivity of up to 0.1 copies [42]. In addition, S ączy ńska V et al established a novel epitope-blocking ELISA (EB-ELISA) for the specific and sensitive detection of anti-HA antibodies against the H5 subtype [43].…”