Detection of avian influenza virus by RAA-CRISPR/Cas13a
10.16303/j.cnki.1005-4545.2024.10.10
- VernacularTitle:基于RAA-CRISPR/Cas13a的禽流感病毒检测方法的建立
- Author:
Xiangyun LE
1
,
2
;
Zhihang FENG
;
Yanli FAN
;
Qiang ZHANG
;
Yicun CAI
;
Wei XIONG
;
Xiang WANG
;
Qingli DONG
;
Jian LI
;
Junxin XUE
;
Yan WANG
Author Information
1. 上海海关动植物与食品检验检疫技术中心,上海 200135
2. 上海理工大学 健康科学与工程学院,上海 200093
- Keywords:
avian influenza virus;
RAA;
CR1SPR/Cas13a
- From:
Chinese Journal of Veterinary Science
2024;44(10):2153-2158,2171
- CountryChina
- Language:Chinese
-
Abstract:
An innovative on-site real-time avian influenza virus(AIV)detection method was estab-lished by integratingrecombinase-aided amplification(RAA)with the clustered regularly inter-spaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)system.After analy-zing 120 sequences of the M gene of avian influenza viruses of different subtypes publicly available on NCBI,the RAA primers and crRNA were designed based on the identified highly conserved segment and used for RAA nucleic acid amplification.After the amplified products were transferred to a CRISPR/Cas13a detection system,the fluorescence values were monitored throughout the re-action process to indicate the results.The sensitivity and specificity of the RAA-CRISPR/Cas13a method were validated using gradient dilutions(106-100 copies/μL)of positive plasmids and sev-en other avian viruses.Fifty clinical samples were tested using this method and compared with the national standard fluorescence RT-PCR method.The results indicated that the detection limit for RAA-CRISPR/Cas13a method was 102 copies/μL,a two-fold improvement over the standard RAA.Specificity assay showed the established method only detected AIV with no cross-reactivity with other seven avian viruses.Compared to the national standard fluorescence RT-PCR method,this method exhibited 100％specificity,95.24％accuracy,and 98.00％consistency in detection of clinical samples.In conclusion,a universal and rapid RAA-CRISPR/Cas13a for detection of AIV was established with the capacity of achieving detection within 60 minutes at 37 ℃,which provides a rapid,sensitive,and specific on-site detection method for AIV.
