Establishment and evaluation of a CRISPR/Cas13a-based method for HBV DNA detection
10.3760/cma.j.cn114452-20241027-00586
- VernacularTitle:基于CRISPR/Cas13a技术检测乙型肝炎病毒DNA方法的建立和评价
- Author:
Yinkang MO
1
;
Zihao FAN
1
;
Yuan TIAN
1
;
Ling XU
1
;
Yaling CAO
1
;
Feng REN
1
Author Information
1. 首都医科大学附属北京佑安医院/北京肝病研究所，北京100069
- Publication Type:Journal Article
- Keywords:
Hepatitis B;
Microbial sensitivity tests;
Molecular detection
- From:
Chinese Journal of Laboratory Medicine
2025;48(4):478-483
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a method for hepatitis B virus (HBV) DNA detection based on recombinase-mediated isothermal amplification (RAA)-clustered regularly interspaced short palindromic repeats and their associated protein 13a (CRISPR-Cas13a).Methods:Through the alignment and screening of HBV DNA sequences, a positive plasmid was constructed, and recombinase-aided amplification (RAA) primers and CRISPR RNA (crRNA) were designed. A method for detecting HBV DNA based on the RAA-CRISPR-Cas13a system was developed, and the specificity and sensitivity were evaluated. Utilizing the CRISPR-Cas13a system, 70 clinical samples from HBV DNA-positive patients with various viral loads collected at Beijing You′an Hospital from 2019 to 2021 were analyzed. The detection results were further compared with those results using real-time quantitative polymerase chain reaction (qPCR).Results:The optimal RAA amplification primers and crRNA were first screened using the RAA-CRISPR-Cas13a method, with the sensitivities for detecting HBV DNA standards and for clinical samples at 1 IU/ml and<10 IU/ml, respectively, demonstrating specificity for HBV DNA detection. Compared with qPCR (the gold standard), the detection consistency between the two methods was 100% (70/70).Conclusion:This study established a method for detecting HBV DNA by integrating recombinase-aided amplification (RAA) technology with CRISPR/Cas13a technology.
