CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

Abstract

Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.

Keywords: CRISPR/Cas12a; Hepatitis B virus (HBV); LAMP; point-of-care detection.

Figure 1

Cas12a-DETECTR assay of HBV DNA. After HBV serum samples were rapidly processed within 10 min, the extracted HBV DNA was amplified by performing LAMP of the target sequence (15–30 min). Cognate binding of the Cas12a–crRNA complex to amplified HBV DNA targets triggered the collateral activity of Cas12a, which cleaved ssDNA reporters (15–30 min). The cleaved ssDNA is visualized by both fluorescence readout (FAM-BHQ) and lateral flow test strips (FITC-Biotin).

Figure 2

Preparation of Cas12a-DETECTR system. (a) HBV genome map shows target and crRNA sequences on the polymerase coding region. (b) The sequence and location of LAMP primers and target sites in the HBV genome. (c) To screen good crRNA, we performed Cas12a assays on 10⁷ copies/μL of HBV plasmids by using crRNA 1 and 2; neg: parallel control without crRNA addition for the same Cas12a assay. (d) To screen the best LAMP primer, after the amplification of different LAMP primers, we performed agarose gel electrophoresis.

Figure 3

Determination of LoD and specificity of Cas12a-DETECTR assay. (a) Fluorescence curves generated by the Cas12a-DETECTR reaction at each dilution. Data were expressed as mean ± SD. from triplicate assays. Negative control (NC) utilized RNase-free water as input instead of HBV DNA dilutions. (b) Comparison of fluorescence values generated after 2 min of Cas12a-DETECTR reaction at each dilution. (c) Fluorescence curves generated by the Cas12a-DETECTR reaction at each LAMP incubation time gradient (HBV concentration is 1 copy/μL). (d) Comparison of fluorescence values generated after 3 min of Cas12a-DETECTR reaction at each LAMP incubation time gradient (HBV concentration, 1 copy/μL). (e) Comparison of lateral flow test strip results at each Cas12a-DETECTR reaction time gradient after 15 min of LAMP incubation (HBV concentration is 1 copy/μL). (f) HBV Cas12a-DETECTR fluorescence readout assays for different hepatitis virus clinical samples. (g) The fluorescence values generated after 30 min of Cas12a-DETECTR reaction for each virus. (h) The sequence alignment of nucleotides in the crRNA 1 target region of the HBV gene with other common human hepatitis viruses, including HAV, HCV, HDV, and HEV. +: indicates a positive sample, -: indicates a negative sample. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Figure 4

Cas12a-DETECTR assay for HBV in 73 clinical samples, which were tested using qPCR and Cas12a-DETECTR (including fluorescence readout and lateral flow test strip readout). Evaluation of Cas12a-DETECTR assay results with qPCR assay results. HBV positivity was defined based on qPCR results. +: indicates a positive sample, -: indicates a negative sample.