CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection

• Published in: Emerging microbes & infections 2023-12, Vol.12 (2), p.2276337-2276337
• Main Authors: Tian, Yuan, Fan, Zihao, Zhang, Xiangying, Xu, Ling, Cao, Yaling, Pan, Zhenzhen, Mo, Yinkang, Gao, Yao, Zheng, Sujun, Huang, Jing, Zou, Huaibin, Duan, Zhongping, Li, Hao, Ren, Feng
• Format: Article
• Language: English
• Subjects: Cohort Studies; CRISPR; CRISPR–Cas13; droplet digital PCR; Hepatitis; Hepatitis B, Chronic; Hepatitis delta virus; Hepatitis Delta Virus - genetics; Humans; Immunoglobulin G; Infections; Prospective Studies; quantitative real-time PCR; recombinase-aided amplification; RNA, Viral - genetics; Sensitivity and Specificity
• ISSN: 2222-1751; 2222-1751
• DOI: 10.1080/22221751.2023.2276337

Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR-Cas13a technology. We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR-Cas13a combined with RT-PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR-Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT-qPCR and RT-ddPCR. For synthetic HDV RNA plasmids, the sensitivity of RT-PCR-CRISPR-based fluorescence assays was 1 copy/μL, higher than that of RT-qPCR (10 copies/μL) and RT-ddPCR (10 copies/μL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/μL, as low as that of RT-qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT-qPCR, RT-ddPCR, RT-PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively. We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.