Establishment of a rapid HIV-1 nucleic acid detection method based on reverse transcription-recombinase aided amplification（RT-RAA）and clustered regularly interspaced short palindromic repeats（CRISPR）-Cas12a
10.3760/cma.j.cn112866-20250910-00195
- VernacularTitle:基于RT-RAA和CRISPR-Cas12a的HIV-1核酸快速检测方法的建立
- Author:
Xin ZHANG
1
;
Pinliang PAN
1
;
Boxue HAN
1
;
Cong JIN
1
Author Information
1. 传染病溯源预警与智能决策全国重点实验室 中国疾病预防控制中心性病艾滋病预防控制中心参比实验室，北京 102206
- Publication Type:Journal Article
- Keywords:
Human immunodeficiency virus（HIV）;
Nucleic acid detection;
Recombinase aided amplification（RAA）;
Clustered regularly interspaced short palindromic repeat
- From:
Chinese Journal of Experimental and Clinical Virology
2025;39(5):623-630
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a novel rapid HIV-1 nucleic acid detection method based on reverse transcription-recombinase aided amplification（RT-RAA）and clustered regularly interspaced short palindromic repeats（CRISPR）technology.Methods:A total of 610 full-length HIV-1 genome sequences reported in China were analyzed，and primers targeting the relatively conserved region of the HIV-1 pol gene and three crRNAs were designed. The crRNAs were validated and screened，and the CRISPR detection system was optimized to establish a “one-pot” HIV-1 RT-RAA/CRISPR assay. Ten recombinant plasmids representing HIV-1 subtypes reported in China were constructed and serially diluted to evaluate the subtype coverage and sensitivity of the assay. The specificity of the assay was evaluated using plasma samples from hepatitis B virus（HBV），hepatitis C virus（HCV）and treponema pallidum（TP）-infected individuals. The clinical performance of the assay was preliminarily evaluated using 45 clinical samples（25 plasma samples from HIV-1 infected individuals and 20 plasma samples from healthy individuals）.Results:The crRNA combination demonstrated higher sequence coverage and higher terminal fluorescence compared to single crRNA. The HIV-1 RT-RAA/CRISPR assay established with the crRNA combination could complete the detection in 30 minutes. This method could detect all 10 reported HIV-1 subtypes in China，with a sensitivity as low as 1-10 copies/μl. No cross-reactivity was observed with HBV，HCV and TP. The results of clinical sample testing were 100% concordant with those of real time fluorescence quantitative RT-PCR assay.Conclusion:This study established a novel HIV-1 RT-RAA/CRISPR nucleic acid detection method with broad subtype coverage，simplicity，rapidity，high sensitivity，and strong specificity. The method shows potential for application in point-of-care testing of HIV-1.
