Analysis of the Advantages and Clinical Validation of Real-Time Fluorescence Nucleic Acid Isothermal Amplification Detection Technology in Detecting Low Viral Load Samples From HBV-Infected Patients

Abstract

Objective 

 This paper examines the advantages and clinical application value of simultaneous amplification and testing (SAT) real-time fluorescent nucleic acid isothermal amplification detection technology for detecting low viral load samples in individuals infected with hepatitis B virus (HBV).

Methods 

 Using reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) as the reference, the detection performance of real-time fluorescence nucleic acid isothermal amplification detection technology (SAT) was evaluated, including linear range, precision, and detection limit. Both methods were used to detect samples, which were the national standard substances of hepatitis B virus ribonucleic acid (HBV RNA). A total of 170 patients with chronic HBV infection were included for methodological comparison. They were divided into a high-level group (serum HBV DNA > 100 IU/mL, n = 111) and a low-level group (serum HBV DNA ≤ 100 IU/mL, n = 59) based on serum HBV DNA levels. The correlation and consistency of the test results from the two methods were evaluated. Additionally, the distribution characteristics of HBV RNA and its correlation with HBV markers were analyzed using data from 1006 patients with chronic HBV infection.

Results 

 Compared with the RT-qPCR method for detecting HBV RNA, the SAT method demonstrated a wider linear range (102-108 copies/mL vs. 103-108 copies/mL), higher precision for low-concentration samples (intra-assay coefficient of variation: 4.23% vs. 12.82%), and a lower detection limit (50 copies/mL vs. 500 copies/mL). In clinical sample testing, the overall detection rate of the SAT method was higher than that of the RT-qPCR method (72.35% vs. 57.64%, P < 0.01), and the detection rate of the SAT method was also higher in the HBV DNA low-level group (50.85% vs. 28.81%, P = 0.007). Large-sample analysis showed that among patients with HBV DNA < 20 IU/mL, 40.4% still had detectable HBV RNA, and the positive rate for HBsAg ≥ 100 IU/mL was 55.5%. Correlation analysis indicated that HBV RNA was moderately positively correlated with HBsAg (r = 0.506) and HBeAg (r = 0.454), and weakly negatively correlated with ALT (r = －0.098) and AST (r = －0.082) (all P < 0.05).

Conclusion 

 The SAT method offers higher sensitivity and stability in detecting low-level HBV RNA. HBV RNA can serve as a serological marker for evaluating viral transcription activity and has clinical application value in managing patients who are HBV DNA-negative or have low HBV DNA levels.

Keywords: Hepatitis B virus, Simultaneous amplification and testing, Clinical trial, Ultra-low limit detection, Covalently closed circular DNA (cccDNA)