A CRISPR-Cas12b-Based Platform for Ultrasensitive, Rapid, and Highly Specific Detection of Hepatitis B Virus Genotypes B and C in Clinical Application

Abstract

Hepatitis B virus (HBV) is one of the most dangerous and prevalent agents that causes acute and chronic liver diseases in humans. Genotyping plays an important role in determining clinical outcomes and response to antiviral treatment in HBV-infected patients. Here, we first devised a CRISPR-based testing platform, termed "CRISPR-HBV," for ultrasensitive, highly specific, and rapid detection of two major HBV genotypes (HBV-B and HBV-C) in clinical application. The CRISPR-HBV employed multiple cross displacement amplification (MCDA) for rapid preamplification and then Cas12b-based detection for decoding the targets. Finally, the detection result was read out with real-time fluorescence and a lateral flow biosensor. The sensitivity of CRISPR-HBV was 10 copies per test. The specificity was one hundred percent, and no cross reactions were observed in other HBV genotypes and pathogens. The whole detection process, including DNA template extraction (15 min), preamplification reaction of MCDA (30 min at 65°C), CRISPR-Cas12b-based detection (5 min at 37°C), and results readout (∼2 min), could be completed within 1 h. The feasibility of the CRISPR-HBV assay for genotyping HBV-B and -C as successfully validated with clinical samples. Hence, the CRISPR-HBV assay has remarkable potential to develop a point-of-care testing for identifying and distinguishing HBV genotypes B and C in clinical settings, especially in resource-scarcity countries.

Keywords: CRISPR; Cas12b; hepatitis B virus; lateral flow biosensor; multiple cross displacement amplification.

FIGURE 1

A schematic illustration of the principle of the CRISPR-HBV system. (A) Schematic illustration of the principle of MCDA with the modified primer. The amplification primer D1 was modified with a PAM site (TTC). After amplification, a CRISPR-Cas12b recognition site was constructed in the target amplicons. (B) Schematic illustration of the CRISPR-Cas12b detection system. Upon recognition of the matching target sequence, the CRISPR-Cas12b complex cleaves a single-stranded DNA reporter molecule. © Sequences and location of the S gene of HBV genotypes B and C used to devise the MCDA primers and gRNAs. The sites of MCDA primers are underlined, and the gRNAs are in boxes. Right arrows and left arrows indicate the sense and complementary sequences which were used in this study, respectively.

FIGURE 2

An outline of the CRISPR-HBV workflow. (A) The CRISPR-HBV RTF assay employs three closely linked steps: DNA extraction (step 1), MCDA (step 2), and CRISPR-Cas12b cleavage and RTF readout (step 3). The whole detection process could be completed within 1 h. (B) CRISPR-HBV–LFB assay employs four closely linked steps: DNA extraction (step 1), MCDA (step 2), CRISPR-Cas12b cleavage (step 3), and LFB readout (step 4). The whole detection process could be completed within 60 min.

FIGURE 3

The schematic of the LFB for visualization of HBV genotype B and C products. (A) The reaction mixtures (1.5 μl) and the running buffer (50 μl) were deposited on the sample pad. (B) The running buffer containing mixtures moved along the LFB owing to capillary action; meanwhile, the dye and streptavidin-coated gold nanoparticles (GNPs) rehydrated in the conjugate region. (C) In the positive sample, the ssDNA reporter molecule (5′-FAM-TTTTTT-Biotin-3′) was trans-cleaved by the activated CRISPR-Cas12b nuclease and the FAM and biotin were separated. Hence, the biotin–streptavidin–GNPs complex was captured by biotin-BSA at the TL; however, in negative outcomes, the ssDNA reporter molecule was not cleaved and was specifically captured by the anti-FAM at the CL. The biotins of the ssDNA reporter molecule bind streptavidin–GNPs for visualization at the CL. (D) Interpretation of the CRISPR-HBV assay results. For positive results, the CL and TL appear on the LFB. When only the CL is observed on the LFB, it indicates negative outcomes. CL: control line; TL: test line.

FIGURE 4

The sensitivity of the CRISPR-HBV assay. LFB and RTF techniques were simultaneously applied for reporting the CRISPR-HBV assay results. LFB (A) and RTF (B) 1–8 represent the HBV genotype B-S plasmid concentrations of 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰, and 1 × 10^-1 copies per reaction and the blank control (DW), respectively. LFB (B) and RTF (D) 1–8 represent the HBV genotype C-S plasmid concentrations of 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰, and 1 × 10^-1 copies per reaction and the blank control (DW), respectively. The LoD of the CRISPR-HBV assay was 10 copies per reaction. CL: control line; TL: test line; “+”: positive; “−”: negative.

FIGURE 5

The specificity of the CRISPR-HBV assay. (A) The specificity of the CRISPR-HBV–LFB assay for HBV genotype B detection. Biosensor 1, HBV genotype B-S plasmid; biosensors 2–9, HBV genotype B agents (clinical samples); biosensors 10–13, HBV genotype B/C agents (clinical samples); biosensor 14, HBV genotype A-S plasmid; biosensor 15, HBV genotype C-S plasmid; biosensor 16, HBV genotype D-S plasmid; biosensor 17, HBV genotype E-S plasmid; biosensor 18, HBV genotype F-S plasmid; biosensor 19, HBV genotype G-S plasmid; biosensor 20, HBV genotype H-S plasmid; biosensor 21, hepatitis C virus (standard substance); biosensor 22, human immunodeficiency virus (standard substance); biosensor 23, human rhinovirus; biosensor 24, adenovirus; biosensor 25, Mycobacterium tuberculosis; biosensor 26, Bordetella pertussis; biosensor 27, Bacillus cereus; biosensor 28, Haemophilus influenzae; biosensor 29, Staphylococcus aureus; and biosensor 30, blank control. CL: control line; TL: test line; “+”: positive; “−”: negative. (B) The specificity of the CRISPR-HBV–LFB assay for HBV genotype C detection. Biosensor 1, HBV genotype C-S plasmid; biosensors 2–9, HBV genotype C agents (clinical samples); biosensors 10–13, HBV genotype B/C agents (clinical samples); biosensor 14, HBV genotype A-S plasmid; biosensor 15, HBV genotype B-S plasmid; biosensor 16, HBV genotype D-S plasmid; biosensor 17, HBV genotype E-S plasmid; biosensor 18, HBV genotype F-S plasmid; biosensor 19, HBV genotype G-S plasmid; biosensor 20, HBV genotype H-S plasmid; biosensor 21, hepatitis C virus (standard substance); biosensor 22, human immunodeficiency virus (standard substance); biosensor 23, human rhinovirus; biosensor 24, adenovirus; biosensor 25, Mycobacterium tuberculosis; biosensor 26, Bordetella pertussis; biosensor 27, Bacillus cereus; biosensor 28, Haemophilus influenzae; biosensor 29, Staphylococcus aureus; and biosensor 30, blank control. CL: control line; TL: test line; “+”: positive; “−”: negative.