Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system

Abstract

Objectives: Undetectable or low-level hepatitis B virus (HBV) DNA and drug resistance mutations in patients may increase the risk of HBV transmission or cause active viral replication and other clinical problems. Here, we established a highly sensitive and practical method for HBV and drug resistance detection using a polymerase chain reaction (PCR) -based CRISPR-Cas13a detection system (referred to as PCR-CRISPR) and evaluated its detection capability using clinical samples. Methods: Specific CRISPR RNAs (crRNAs) are designed for HBV DNA detection and YMDD (tyrosinemethionine-aspartate-aspartate) variant identification. The HBV DNA was detected in 312 serum samples for HBV diagnosis using quantification PCR (qPCR) and PCR-CRISPR. Additionally, 424 serum samples for YMDD testing were detected by qPCR, direct sequencing, and our assay. Results: Using PCR-CRISPR, one copy per test of HBV DNA was detected with HBV-1 crRNA in 15 min after PCR amplification. Consistent results with qPCR were observed for 302 samples, while the remaining 10 samples with low-level HBV DNA were detectable by PCR-CRISPR and droplet digital PCR but not by qPCR. PCR-CRISPR diagnosed all 412 drug-resistant samples detected by the YMDD detection qPCR kit and direct sequencing, as well as the other 12 drug-resistant samples with low-level HBV DNA undetectable by qPCR and direct sequencing. Conclusions: We developed a novel PCR-CRISPR method for highly sensitive and specific detection of HBV DNA and drug resistance mutations. One copy per test for HBV DNA and YMDD drug resistance mutations could be detected. This method has wide application prospects for the early detection of HBV infection, drug resistance monitoring and treatment guidance.

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

“…With the development of CRISPR/Cas-based systems for nucleic acid detection, rapid, accurate, and portable point-of-care detection of pathogens has become a reality. Although the highly sensitive detection of HBV based on PCR-Cas13a has been achieved, it still requires complex nucleic acid extraction of clinical samples, specialized PCR amplification, and fluorescence collection of Cas13a by the qPCR system, thus limiting the efficiency of detection and point-of-care testing [ 24 ].…”

Droplet digital PCR of viral ‎DNA/RNA, current progress, challenges, and future perspectives

“…183,184 Due to the acceptable sensitivity of the test, ddPCR is implemented with automation in support of multiple upstream and downstream process development efforts for influenza vaccine manufacturing. 185 In the dengue virus workflow, the RT-ddPCR assay will provide absolute quantification of all four serotypes in a single multiplex assay without the need for a standard curve. The assay also required less time and reagent use and reduced technical error probability.…”

CRISPR/Cas System: A Potential Technology for the Prevention and Control of COVID-19 and Emerging Infectious Diseases

“…So the identification of mutant strains based on high-specificity CRISPR/Cas technology has great significance. Although no researchers have yet performed CRISPR/Cas-based detection of the SARS-COV-2 mutation site, some scientists have successfully used Cas13a to detect the HBV drug resistance site rt204 (Gootenberg et al, 2017;Wang S. et al, 2020).…”