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.

Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System

“…The ERA amplification only took 15 min, the incubation time of the dipstick detection system was 15 min, and the fluorescence system reached a plateau in approximately 20 min; therefore, the ERA/CRISPR-Cas12a dual system only took 30 min to detect M. pneumoniae. Furthermore, M. pneumoniae DNA extraction could be completed within 25 min, and the time from samples to results of M. pneumoniae was approximately 1 h, which was generally consistent with the reaction time of the CRISPR-HBV system (Chen et al, 2021) and Cas12a-DETECTR assay (Ding et al, 2021), and was much lower than the 3-h detection time of Cas12a/gRNA trans-cleavage fluorescence assay for MTB detection (Xu et al, 2020). In general, the ERA/CRISPR-Cas12a dual system fulfills the need for rapid nucleic acid detection.…”

Development of CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings

“…In the CRISPR/Cas system, the CRISPR/Cas effector proteins (e.g., Cas12a, Cas12b, Cas13a, and Cas13b) are specifically navigated by guide RNA (gRNA) to the target to form the complex (namely, target/Cas nuclease/gRNA complex), which can nonspecifically and indiscriminately cleave nearby nontarget single-stranded DNA (ssDNA) and/or ssRNA reporters when the trans -cleavage activity of Cas nuclease (i.e., collateral cleavage activity) is activated ( 29 – 34 ). Of the various Cas nucleases, the most widely used, Cas12a, Cas12b, and Cas13a, have been successfully combined with isothermal amplification techniques ( 33 – 35 ), and a series of CRISPR/Cas12a-, CRISPR/Cas12b-, and CRISPR/Cas13a-based nucleic acid detection techniques have been devised and used in past studies, such as DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter), SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), and MCCD (multiple cross displacement amplification CRISPR/Cas12a detection) ( 29 , 36 , 37 ). These CRISPR/Cas-based assays greatly shorten detection time and improve detection sensitivity, providing an efficient diagnostic platform for target pathogen detection.…”

A CRISPR-Cas12a—Based platform for ultrasensitive, rapid, and highly specific detection of Mycoplasma pneumonia in clinical application

“…Hitherto, various isothermal amplification techniques coupled with different biosensing systems have been developed for MP infection diagnosis, such as MCDA combined with nanoparticle-based lateral flow biosensor (LFB) ( Wang et al, 2019 ), LAMP combined with LFB ( Xiao et al, 2022 ), and recombinase polymerase amplification (RPA) combined with real-time fluorescent probe ( Jiang et al, 2022 ), incarnating the advantages of sensitivity, specificity, simplicity and rapidity. Discovery of the CRISPR-Cas system provided a superior alternative for nucleic acid detecting platform for the high specificity (single base pair specificity) and sensitivity (only several copies) ( Zhu et al, 2021 ), and the CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13 systems had been applied for detection of a variety of pathogens, such as hepatitis B virus ( Chen et al, 2021 ) and SARS-CoV-2 ( Zhu et al, 2021 ). In this report, the CRISPR-Cas12a system was first applied as the biosensing platform for MP detection for its highly sensitive, specific, and accurate.…”