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.

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

“…Detection of HIV-1 termed as Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN) can recognize target DNA concentrations at least to 10 nM within 1 h, without the requirement of pre-amplification steps ( Nouri et al, 2020 ). A report detecting HBV based on DETECTR showed an LoD of 1 copy/μl within 13 min using fluorescent readout; however, the LoD of lateral flow test strip technique costing 20 min was not shown ( Ding R. et al, 2021 ). Utilizing lateral flow, a Cas12a-based biosensor was designed for the detection of Epstein–Barr virus (EBV), achieving a sensitivity of 7.1 × 10 –14 mol/L (approximately 42,000 copies per μl) ( Yuan et al, 2020 ).…”

“…Currently, clustered regularly interspaced short palindromic repeats (CRISPR) based biosensors have been developed as powerful tools for nucleic acid sensing and widely applied to the rapid diagnosis of infectious pathogens ( Kellner et al, 2019 ; Qin et al, 2019 ; Broughton et al, 2020 ; Ding R. et al, 2021 ; Song F. et al, 2021 ; Chen et al, 2021c ; Escalona-Noguero et al, 2021 ; Ge et al, 2021 ) and the detection of DNA or miRNAs associated with diseases such as cancer ( Li B. et al, 2021 ; Wang et al, 2021c ; Chen Y. et al, 2021 ). These biosensors rely on the CRISPR systems containing CRISPR-associated proteins (Cas) with nonspecific endonuclease activity to efficiently cleave specific targets via guide RNAs (gRNAs) ( Cong et al, 2013 ; Mali et al, 2013 ; van der Oost, 2013 ).…”

Potential of the CRISPR‐Cas system for improved parasite diagnosis

“…[70] The advantages of using a LAMP-Cas12a diagnostics system were further supported by a report of a rapid and accurate POC assay for hepatitis B (HBV), achieving a limit of HBV detection of 1 copy/µL using a fluorescent readout or lateral flow test strips within 13 and 20 minutes, respectively, showing 100% sensitivity and comparable specificity with qPCR. [71] The LAMP coupled CRISPR-Cas12a module was also extended for the sensitive detection of plant DNA viruses providing easy-to-interpret visual readouts in ∼1 hour using a simple, low-cost fluorescence visualizer, making it suitable for POC applications. [72] Overall, the successful development of field-applicable CRISPR-Cas12 based diagnostics highlights the potential of extending this novel technology to detect other infectious diseases, including parasitic infections.…”

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

“…Once Cas12a/crRNA bound to an activator (ssDNA or dsDNA) that has complementary base-pairing to the guide crRNA, the trans- acting cleavage of Cas12a is initiated and then capable of nonspecific cleavage of ssDNA (single-stranded DNA) ( Chen et al, 2018 ). When combined with a nonspecific ssDNA reporter, Cas12a has been used to detect nucleic acids reliably ( Bai et al, 2019 ; Ding et al, 2021 ; Gong et al 2021a , 2021b ; Ma et al, 2021 ; Wang et al, 2021b ). By introducing an additive mismatch in crRNA, the CRISPR/Cas12 system can even be used for single-nucleotide polymorphism (SNP) genotyping with single-base specificity ( Chen et al, 2021 ; Huang et al, 2021 ; Lee et al, 2020 ; Lee Yu et al, 2021 ; Meng et al, 2021b ).…”