Rapid visual detection method for duck astrovirus type 2 based on RPA-CRISPR/Cas13a-LFD
10.16303/j.cnki.1005-4545.2025.07.03
- VernacularTitle:基于RPA-CRISPR/Cas13a-LFD的鸭星状病毒2型快速可视化检测方法的建立
- Author:
Shuhai HE
1
;
Mengxiao TAO
;
Luyao WANG
;
Defang ZHOU
;
Jing ZHOU
;
Ziqiang CHENG
;
Li HUANG
Author Information
1. 信阳农林学院动物科技学院,河南信阳 464000;山东农业大学动物科技学院,山东 泰安 271018
- Publication Type:Journal Article
- Keywords:
duck astrovirus type 2;
recombinant enzyme polymerase amplification;
CRISPR/Cas13a;
lateral flow dipstick;
nucleic acid detection
- From:
Chinese Journal of Veterinary Science
2025;45(7):1372-1377
- CountryChina
- Language:Chinese
-
Abstract:
To achieve efficient and rapid detection of duck astrovirus type 2(DAstV-2),RPA prim-ers and crRNA were designed and synthesized based on the conserved sequence of the ORF2 gene of DAstV-2.A detection method for DAstV-2 was constructed,integrating RPA nucleic acid ampli-fication,LwCas13a cleavage,and colloidal gold lateral flow dipstick visualization.The specificity,sensitivity,and concordance of this detection method were evaluated.The experimental results showed that the detection limit for the DAstV-2 recombinant plasmid standard was 1.2×101 cop-ies/μL,which is superior to the conventional RT-PCR method.The method can specifically detect DAstV-2 pathogenic nucleic acids without cross-reactivity with DAstV-1,DAstV-3,DAstV-4,duck plague virus(DEV),and duck tembusu virus(DTMUV).When testing liver tissue samples from ducks suspected of being infected with DAstV-2,the results obtained using this method were com-pletely consistent with those from real-time quantitative PCR,with a 100％concordance rate.How-ever,this method is simpler and faster to perform.The research indicates that the established RPA-CRISPR/Cas13a-LFD detection system has high sensitivity,strong specificity,and high accuracy,capable of completing rapid visual detection of DAstV-2 nucleic acids within 1 h at a constant tem-perature of 37 ℃,providing a new technical platform for the rapid diagnosis of DAstV-2.
