Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Summary

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.

Abstract

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4^th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

Introduction

Hepatitis B virus (HBV) is a partially double-stranded DNA virus from the genus Orthohepadnavirus and the Hepadnaviridae family¹. It can trigger a chronic infection that persists throughout one's life, potentially leading to liver cirrhosis and hepatocellular carcinoma^2,3,4. According to the World Health Organization (WHO), an estimated population of 296 million people were living with chronic hepatitis B infection in 2019, and 1.5 million people were newly infected each year⁵.

The identification and measurement of HBV DNA in serum or plasma samples serve as a valuable method for detecting individuals with an ongoing hepatitis B infection, assessing the efficacy of antiviral treatment, and predicting the probability of treatment success^{6,7,8,9,10,11}. High viral load is associated with an increased risk of liver disease progression, including cirrhosis and liver cancer^12,13. Hence, precise measurement of viral load is crucial for tracking the progression of HBV infection and informing decisions regarding treatment.

Quantitative real-time PCR assays have higher sensitivity, broader dynamic range, and more accurate quantification of HBV DNA than conventional PCR techniques^14,15,16,17. Several commercial real-time PCR-based molecular diagnostic kits for the quantitation of HBV DNA in serum or plasma samples are available in the market. Here, we describe a detailed workflow for the detection and quantification of HBV DNA in human serum or plasma using a commercially available, IVD-marked real-time PCR-based kit. The kit is a widely used^18,19, low-cost, yet highly sensitive assay, and its performance characteristics are comparable with other commercially available CE-marked kit(s)¹⁸. Apart from the HBV target region amplification, an endogenous internal control gene is also included in the kit to verify the quality of samples, quality of extracted DNA, PCR amplification, and possible PCR inhibition.

Protocol

This study did not involve any human participants or clinical samples. The only biological material used was the 4^th WHO International Standard for HBV DNA for nucleic acid test (NIBSC code 10/266). This standard is a publicly available reference material that does not contain any personal information or identifiable data. As such, no ethical approval was required for this study.

1. DNA Extraction

1. Extract the viral DNA from human serum or plasma sample. Store the extracted DNA at -20 °C until further use in PCR.
   ​NOTE: The recommended volume of serum or plasma for DNA extraction is 500 µL, and the elution volume is 50 µL. In this study, the viral DNA was extracted using a viral nucleic acid extraction kit (Table of Materials).

2. Real-time PCR

1. Thaw all the reagents (Table 1) of the real-time PCR kit at room temperature (RT; 15-25 °C) before starting the PCR. When thawed, mix the components by pulse vortexing for 10 s at a moderate speed and centrifuge at 8,000 × g for 15 s at RT. Keep all the thawed components on a cooling block.
2. Reaction preparation
   1. Prepare a PCR mix according to Table 2. Calculate the volume of the reagents to be added based on the number of samples, standards, and no template control.
      NOTE: When preparing the PCR mix, at least 10% extra volume of each reagent should be added (e.g., if 10 reactions are required, calculate for 11 reactions).
   2. Pipet 15 µL of the above-prepared PCR mix into each PCR tube. Then, add 15 µL of the extracted sample DNA. Add 15 µL of at least one of the standards for HBV (HBV Standard 1-5) as a positive control (PC) for detecting HBV DNA and 15 µL of PCR-grade nuclease-free water as a negative control (NC). To generate a standard curve required for quantitation of the HBV DNA, use all 5 quantitation standards supplied with the kit (HBV Standard 1-5) for each PCR run.
   3. Close the tubes, mix the components by pulse vortexing for 10 s at a moderate speed, and centrifuge at 8,000 × g for 15 s at RT before proceeding to the thermal cycler. Ensure no bubbles are in the reaction mix, as it may interfere with fluorescence detection.
3. Program setup
   1. Program the thermal cycler using the cycling conditions as recommended in Table 3.
4. Channel/Fluorophore Selection
   1. Select the fluorescent dyes for commonly used real-time PCR platforms, as mentioned in Table 4.
5. Data analysis
   1. After the run is completed, analyze the amplification plot on a linear scale.
   2. Threshold setting
      1. Set the threshold within the exponential phase of a PCR reaction. Recommended threshold values for the kit are mentioned in Table 5. Be consistent with the threshold setting. Once the optimal threshold for the assay is determined, use it consistently for all the samples for a particular PCR machine. This will help to ensure that the results are accurate and reproducible.
         ​NOTE: If the threshold is set too low, the signal may be below the noise level, and the Ct value will be unreliable. If the threshold is set too high, the signal may be in the plateau phase of the reaction, where the amplification is not as efficient, and the Ct value may be inaccurate.
   3. Cutoff
      1. Run the kit for 40 amplification cycles. Do not consider any amplification beyond 38 cycles for any interpretation.
   4. Qualitative result analysis
      1. Use the kit for qualitative detection of HBV DNA. Use Standard 2 as PC with expected Ct values at 20 ± 2 for HBV and 24 ± 3 for IC.
      2. Ensure no template control (NTC) does not exhibit any amplification for both HBV and internal control (IC) targets. If an amplification reaction occurs in the NTC reaction, then sample contamination may have occurred.
      3. Since the assay cutoff is 38, consider any amplification before 38 Ct for the HBV target as an HBV-positive sample. When all controls meet the above-stated requirements, consider a specimen following the interpretations mentioned in Table 6.
   5. Quantitative result analysis
      1. Use the set of 5 quantification standards of known concentrations for HBV-specific DNA that are calibrated against the 4th WHO International standard for HBV DNA for nucleic acid amplification techniques (NAT)²⁰ (Table 7).
      2. Employ these standards to generate a standard curve. Utilize the standard curve to quantify the HBV DNA of an unknown sample.
         NOTE: The real-time PCR software will generate a standard curve using the Ct values obtained for the HBV targets of all 5 standards and their corresponding concentrations in international units per microliter (IU/µL).
      3. Only interpret the values for unknown samples if the slope of standards falls between -3.1 and -3.6, the R² value is between 0.99 and 1.0, the PCR efficiency is between 90%-110% (0.9-1.1), and there is no amplification in the negative control.
      4. The software will calculate the concentration of each HBV-positive sample in international units per microliter (IU/µL). To calculate the viral load (conversion of IU/µL to international units per milliliter [IU/mL]), use the following equation:
         Result (IU/mL) = (Result [IU/µL] x Elution Volume [µL])/ Sample Volume (mL)
         NOTE: The 4^th WHO International Standard for HBV DNA, NIBSC code 10/266, was extracted for viral DNA purification from 0.5 mL plasma, eluted the purified viral DNA into 50 µL of elution buffer and used with the PCR kit as a reference sample along with the 5 HBV standards and NTC. The HBV viral load of the reference sample (NIBSC code 10/266) was calculated as (6659 IU/µL x 50 µL)/0.5 mL = 6,65,900 IU/mL.

Results

A schematic diagram of the workflow to detect and quantify HBV DNA from human plasma/serum is shown in Figure 1. An amplification plot of Standard 2 (used as a PC) and NTC for both HBV and IC is shown in Figure 2. Figure 3 shows the amplification curves for an HBV-positive sample, an HBV-negative sample, and a sample with PCR inhibition. The amplification curve, Ct value for HBV, and obtained HBV concentration in international units...

Discussion

NIBSC code 10/266 has been assigned a unit of 9,55,000 IU/mL when reconstituted in 0.5 mL of nuclease-free water as per the supplier information²¹. This can be considered a high-load HBV positive sample. The HBV viral load determined by the viral load kit used in this study was 6,65,900 IU/mL (Figure 4). As the DNA extraction efficiency of any DNA extraction kit is not 100%, some DNA is often lost during the purification process. Although the viral load determined by ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4th WHO International Standard for hepatitis B virus DNA	NIBSC	NIBSC code: 10/266	The 4th WHO International Standard for hepatitis B virus (HBV) DNA, NIBSC code 10/266, is intended to be used for the calibration of secondary reference reagents used in HBV nucleic acid amplification techniques (NAT).
ABI real-time PCR machines	ThermoFisher Scientific	ABI 7500; QuantStudio 5	This is a real time PCR machine
HBV, HCV and HIV Multiplex 14/198	NIBSC	NIBSC code: 14/198	This is a very low positive triplex reagent for NAT assays containing hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus-1 (HIV-1)
QIAGEN real-time PCR machine	Qiagen	Rotor-Gene Q	This is a real time PCR machine
TRUPCR HBV Viral Load kit	Kilpest India Limited	3B294	This is a real time PCR based kit used in this study for the HBV DNA detection and viral load determination
TRUPCR Viral Nucleic Acid Extraction Kit	Kilpest India Limited	3B214	This is a DNA extraction kit used for the extraction of HBV viral DNA from NIBSC:10/266 and NIBSC:14/198