Detection of H5N6 and H9N2 subtypes of avian influenza viruses with reverse transcription-recombinase polymerase amplification combined with CRISPR-Cas13a
10.3969/j.issn.1002-2694.2025.00.034
- VernacularTitle:CRISPR-Cas13a蛋白结合RT-RPA技术检测H5N6和H9N2亚型禽流感病毒
- Author:
Jing-jing WU
1
;
Yu-wei WENG
1
;
Zhi-miao HUANG
1
;
Hong-bin CHEN
1
Author Information
1. 福建省疾病预防控制中心,福建省人兽共患病研究重点实验室,福州 350012
- Publication Type:Journal Article
- Keywords:
CRISPR-Cas13a;
recombinase polymerase amplification;
avian influenza viruses;
nucleic acid detection
- From:
Chinese Journal of Zoonoses
2025;41(3):235-242
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study was to establish a rapid,highly sensitive,and specific nucleic acid detection method for the H5N6 and H9N2 avian influenza virus(AIV)subtypes by using reverse transcription-recombinase polymerase amplification(RT-RPA)combined with clustered regularly interspaced short palindromic repeats(CRISPR)-Cas13a proteins.The conserved regions were selected to design specific RT-RPA primers and crRNA sequences of H5,H6,H9,and N2 genes.RT-RPA tech-nology combined with CRISPR-Cas13a detection was used to evaluate the sensitivity and specificity of AIV nucleic acid detec-tion.The detection was performed on avian influenza environmental samples and compared with the results of fluorescence quantitative RT-PCR,to evaluate the effectiveness of the RT-RPA technology combined with CRISPR-Cas13a.AIV H5,H9,and N2 subtypes were detected with a sensitivity as high as 1 copy/μL,and AIV N6 subtypes were detected with a sensitivity of 10 copies/μL.Plasmid samples with differing copy numbers showed fluorescence under blue LED transillumination.The four AIV subtypes showed high specificity and did not cross-react with the other AIV subtypes.The detection of avian influenza ex-ternal environmental samples containing AIV H5,N6,and H9 subtypes was consistent with the results of fluorescence quanti-tative RT-PCR,with 100％accuracy.For AIV N2 subtypes,one additional negative sample was detected with 97.9％accuracy.The established RT-RPA technology combined with CRISPR-Cas13a detection enabled sensitive,specific visual detection of AIV H5N6 and H9N2 subtypes.This study provides a new nucleic acid detection method for AIV surveillance and subtype clas-sification.
