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Updated: Jun 25, 2025
Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection
Published on: December 15, 2023
CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic
Haipo Xu1, Gengping Lin2, Ronghua Chen3
1The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, 350025, PR China; Mengchao Med-X Center, Fuzhou University, Fuzhou, 350116, PR China; College of Chemical Engineering, Fuzhou University, Fuzhou, 350116, PR China.
Insights
A new CRISPR/Cas12b-assisted assay offers rapid, sensitive detection of hepatitis B virus (HBV) DNA. This point-of-care test is ideal for screening chronic hepatitis B (CHB) in resource-limited settings.
Area of Science:
- Molecular diagnostics
- Biotechnology
- Infectious disease detection
Background:
- Chronic hepatitis B (CHB) causes high mortality, with millions undiagnosed annually.
- Existing PCR-based assays are not suitable for high-burden, resource-limited settings.
- CRISPR/Cas systems offer sensitive and specific point-of-care testing (POCT) potential.
Purpose of the Study:
- To develop a rapid, sensitive, and precise method for hepatitis B virus (HBV) DNA quantification.
- To create a point-of-care diagnostic assay suitable for resource-limited settings.
- To address the urgent need for effective HBV detection methods.
Main Methods:
- A one-pot, one-step CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) assay was developed.
- Optimization of reaction buffer, enzyme concentration, reporter, temperature, and additives.
- Validation using 236 clinical samples compared to real-time quantitative PCR.
Main Results:
- The assay quantifies HBV DNA within 1 hour with a limit of detection of 25 copies/mL.
- Demonstrated 99.00% sensitivity and 100.00% specificity in clinical samples.
- Showed no cross-reactivity with other blood-borne pathogens.
Conclusions:
- The developed CRISPR/Cas12b-LAMP assay is a convenient and reliable point-of-care diagnostic tool.
- It significantly improves HBV diagnosis capabilities, especially in resource-limited areas.
- The assay is advantageous for rapid CHB screening and evaluating therapeutic efficacy.
Background:
Currently, millions of people suffer from undiagnosed chronic hepatitis B (CHB) infection each year, which leads to high mortality rates attributed to cirrhosis and hepatocellular carcinoma. Previously reported assays, such as PCR-based assays, have limitations in terms of convenient for CHB screening in high-burden regions and resource-limited settings. Recently, diagnosis based on CRISPR/Cas, which has been considered as a potential method of point-of-care test (POCT) in resource-limited settings, offers a significant advantage in terms of high sensitivity and specificity. Therefore, there is an urgent need for the hepatitis B virus (HBV) detection utilizing CRISPR/Cas system.
Results:
We have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR.
Significance:
The utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.
