Journal Article

Rapid detection of avian influenza virus based on CRISPR-Cas12a

Ma, Yue, Zhou, Xu, Deng, Guohua, Wang, Xiurong, Wang, Siwen, Li, Yanbing, Shi, Jianzhong

2023

Overview

Background Avian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations. Methods The Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1–H16 subtype 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 that 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 agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field. Conclusion An RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Acids / Affinity chromatography / Animals / Applications of CRISPR-Cas and genome editing techniques for established and emerging infectious diseases / Aquatic birds / Avian flu / Avian influenza / Avian influenza virus / Avian influenza viruses / Avian orthoavulavirus 1 / Biomedical and Life Sciences / Biomedicine / Birds / blue light / Bronchitis / CRISPR / CRISPR-Cas Systems / CRISPR/Cas system / cross reaction / Cross-reactivity / Dengue fever / Detection / detection limit / Detectors / Diagnosis / Enzymes / Equipment and supplies / Fluorescence / Genes / Identification and classification / Infectious bronchitis virus / Infectious bursal disease virus / Influenza / Influenza A virus / Influenza in Birds - diagnosis / Laboratories / Laboratory equipment / Newcastle disease / Newcastle disease virus - genetics / NP gene / Pathogens / Plasmids / point-of-care systems / Polyacrylamide / polyacrylamide gel electrophoresis / Polymerase chain reaction / Poultry / Protein expression / Proteins / rapid methods / Real-Time Polymerase Chain Reaction - methods / Recombinase / recombinase polymerase amplification / reverse transcriptase polymerase chain reaction / Reverse transcription / RNA / RNA viruses / RT-RPA / Sensitivity and Specificity / Sodium lauryl sulfate / species / Sulfates / Surface active agents / Testing / Virology / Viruses