CRISPR-Cas13a-based detection method for avian influenza virus

Frontiers in Microbiology

Microbiology

Xuexia Wen¹  Zhaomeng Shan¹  Zhe Liu¹  Yan Li¹  Yining Liu¹  Yuhan Wu¹  Yixin Wang¹  Jiaxing Zhan¹  Xiurong Wang²  Yanbing Li² 

[1] Key Laboratory of Livestock Infectious Diseases, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China;State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China;

关键词: avian influenza virus;    recombinase polymerase amplification;    CRISPR-cas13a;    lateral flow dipstick assay;    universal detection;   

DOI  :  10.3389/fmicb.2023.1288951

 received in 2023-09-05, accepted in 2023-09-27,  发布年份 2023

来源: Frontiers

【 摘 要 】

Avian influenza virus (AIV) causes huge losses to the global poultry industry and poses a threat to humans and other mammals. Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. Here, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a based platform was developed to detect AIV. This novel method was developed to specifically detect H1–H16 subtypes of AIV with fluorescence and lateral flow-based readouts and exhibited no cross-reactivity with Newcastle disease virus, avian infectious bronchitis virus, or infectious bursal disease virus. The limit of detection was determined to be 69 and 690 copies/μL using fluorescence and lateral flow as readouts, respectively. The developed assay exhibited 100% consistency with quantitative real-time polymerase chain reaction in detecting clinical samples. The heating of unextracted diagnostic samples to obliterate nuclease treatment was introduced to detect viral RNA without nucleic acid extraction. Single-step optimization was used to perform reverse transcription, recombinase polymerase amplification, and CRISPR-Cas13a detection in a tube. These advances resulted in an optimized assay that could specifically detect AIV with simplified procedures and reduced contamination risk, highlighting the potential to be used in point-of-care testing.

【 授权许可】

Unknown   
Copyright © 2023 Wu, Zhan, Shan, Li, Liu, Li, Wang, Liu, Wen and Wang.