Rapid detection of Avian Influenza Virus based on CRISPR-Cas12a

Abstract

Abstract Background: Avian influenza(AI) refers to the disease caused by infection with avian influenza viruses(AIV). These viruses naturally spread among wild aquatic birds worldwide and can infect domestic poultry, other birds, and animal species. At present, real-time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR) is mainly used to detect the presence of pathogens, which has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits the point-of-care testing (POCT). A rapid, reliable, non-lab equipment reliant, sensitive, and specific diagnostic test is urgently needed in the field of clinical rapid detection and diagnosis. Methods: In this study, Cas12a protein was purified using affinity chromatography with a Ni-Agarose resin and observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific CRISPR-RNA (crRNA) and primers targeting the M and NP genes of AIV were designed and screened out. By combining reverse transcription recombinase polymerase amplification(RT-RPA) with the Cas12a/crRNA trans-cleavage system, the detection system through fluorescence readouts under blue light or using lateral flow strips was established. The sensitivity assays were carried out using a 10-fold dilution series of the plasmids and RNA of M and NP genes as the templates. The specificity of this method was determined by using H1~H16 subtypes AIVs and other avian pathogens such as Newcastle Disease Virus (NDV), Infectious Bursal Disease Virus (IBDV) and Infectious Bronchitis Virus (IBV). Results:The results showed that the method was able to detect AIV and the detection limit can reach 6.7 copies/μL and 12 copies/μL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreeability by comparing with the rRT-PCR and virus isolation in detecting samples from poultry. This portable and accurate method holds great application potential for detecting AIV in the field. Conclusion: In summary, a RT-RPA/CRISPR method was developed for the rapid and sensitive detection of AIV. The new system presents a good potential to be an accurate, user-friendly, inexpensive platform for point-of-care testing applications.