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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.

Representative 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 per microliter (IU/µL) for the NIBSC code 10/266 are shown in Figure 4. Amplification curves for the HBV target of all the 5 HBV standards of the kit and a representative standard curve for the same are shown in Figure 5. The slope of the standard curve is -3.361, R² is 0.99996, and efficiency is 0.98. Amplification curves for the IC target of all 5 HBV standards are shown in Figure 6.

Figure 1: Schematic diagram of the workflow to detect and quantify HBV DNA from human plasma/serum samples. Extract viral DNA from human serum/plasma sample of HBV suspected individual. Prepare the PCR mix by adding each PCR component except the template DNA/ standard(s). Dispense it into PCR tubes/wells. Add the extracted viral DNA/ standard(s) as the template DNA, and close the PCR tubes. Load it into a real-time PCR machine, start the PCR run, and analyze the result after run completion. Please click here to view a larger version of this figure.

Figure 2: Amplification plot of Standard 2 (used as a PC) and NTC. An amplification plot of HBV standard 2 is shown here for both the HBV and IC targets. For qualitative result analysis, Standard 2 can be used as a positive control. For NTC, no amplification was observed for either of the targets. Please click here to view a larger version of this figure.

Figure 3: Amplification plots for HBV positive sample, HBV negative sample, and a sample with PCR inhibition. Amplification plots for three different samples: HBV positive (Case 1), HBV negative (Case 2), and a sample containing PCR inhibitors (Case 3) are shown here. In the case of the HBV positive sample, both the targets are amplified, whereas only IC was amplified for the HBV negative sample, and no amplification for the HBV target was observed as expected. Neither HBV nor IC was amplified for Case 3, which means PCR inhibition occurred due to the PCR inhibitors present in the DNA sample used as the PCR template. Please click here to view a larger version of this figure.

Figure 4: Amplification curve, Ct value for HBV, and obtained HBV concentration (IU/µL) for the NIBSC code 10/266. Extracted viral DNA of NIBSC code 10/266 was run along with the 5 HBV standards and NTC. The amplification curve for the HBV target is shown for the NIBSC code 10/266 sample in the upper panel. Its Ct value for HBV is 18.94, and the calculated concentration is 6659 IU/µL, shown in the lower panel. Please click here to view a larger version of this figure.

Figure 5: Amplification curves for the HBV target of all 5 HBV standards and the standard curve. Amplification curves for the HBV target (green channel) of all 5 HBV standards are shown in the upper panel. A standard curve along with its slope (3.361), R² value (0.99996), and PCR efficiency (0.98) are shown in the lower panel. Please click here to view a larger version of this figure.

Figure 6: Amplification curves for the IC target of all 5 HBV standards. Amplification curves of 5 HBV standards for the IC (yellow channel) are shown here. As expected, they look the same with similar Ct values. Please click here to view a larger version of this figure.

Figure 7: Amplification curves for the HBV target of all 5 HBV standards and NTC were run in triplicate. The amplification plots of three replicates for the HBV target (Green channel) of the 5 HBV standards show similar results. Please click here to view a larger version of this figure.

Figure 8: Amplification curves for the HBV target of NIBSC code 10/266, NIBSC code 14/198, and its five dilutions. The amplification curves for the HBV target (green channel) of the reference samples NIBSC code 10/266, NIBSC code 14/198, and its five dilutions (1.25 IU/mL, 2.5 IU/mL, 5 IU/mL, 25 IU/mL, and 50 IU/mL) are shown here. No amplification was detected for 1.25 IU/mL, while other dilutions showed proper amplification curves before 38 Ct. Please click here to view a larger version of this figure.

Reagent	Description
Multiplex master mix	PCR buffer, Hot Start Taq DNA polymerase, and other PCR components
HBV primer probe mix	Primers and probe mix for HBV detection
Endogenous IC primer probe mix	Primers and probe mix for endogenous internal control (IC) detection
Standards for HBV	·  HBV Standard 1
	·  HBV Standard 2
	·  HBV Standard 3
	·  HBV Standard 4
	·  HBV Standard 5
Negative Control	PCR grade nuclease-free water

Table 1: Reagents supplied with the real-time PCR kit and their description

Reagent	Volume per reaction
Multiplex master mix	11 µL
HBV primer probe mix	2 µL
Endogenous IC primer probe mix	2 µL
Total reaction volume	15 µL

Table 2: Volume of each reagent to be added per reaction for PCR mix preparation

Step	Temperature (°С)	Time	Data collection	Cycles
Initial Denaturation	94	10 min	–	1
PCR cycling	94	15 s	–	40
	55	60 s	On
	72	15 s	–

Table 3: PCR cycling conditions

Target	Rotor-Gene Q*	CFX 96	ABI real-time PCR#
HBV	Green	FAM	FAM
IC	Yellow	HEX	VIC
*Auto-Gain Optimisation Setup- select 'Perform Optimisation Before 1st Acquisition’
#Only for Applied Biosystems’ real-time PCR machines, select ROX as the ‘Passive Reference’ dye during plate setup, as the master mix of the kit contains ROX

Table 4: Dye selection for different real-time PCR platforms

Real Time PCR Instrument	Dyes	Threshold value range§
ABI real-time PCR¶	FAM	0.2–0.25
	VIC	0.05–0.12
Bio-Rad CFX-96†	FAM	100–800
	HEX	50–400
Rotor-Gene Q	Green	0.015–0.03
	Yellow	0.01–0.02
§An absolute threshold value varies from instrument to instrument depending upon instrument’s age, model and calibration status
¶These threshold values are applicable when ROX is selected as the ‘Passive Reference’ dye
†Relative fluorescence is increased by about 2 to 5-fold when Bio-Rad's white PCR tubes are used instead of clear tubes. So, the threshold value should be determined accordingly

Table 5: Recommended threshold values for different real-time PCR platforms

Sample type	Case	Amplification Signal in		Result Interpretation
		FAM/Green	HEX/VIC/Yellow
Control	Standard 2/PC	Yes (20 ± 2)	Yes (24 ± 3)	Standard 2/PC is working properly
	NTC	No	No	NTC is working properly
Sample	1	Yes	Yes / No*	HBV specific DNA detected
	2	No	Yes	HBV specific DNA not detected. Sample does not contain detectable amount of HBV specific DNA
	3	No	No	Possible inhibition of PCR, dilute the DNA sample (1:10) / re-extract DNA from the original sample and repeat the PCR
*Detection of the Internal Control is not required when the HBV target is detected. High HBV DNA load in the sample can lead to reduction or absence of Internal Control signal

Table 6: Result interpretation of positive, negative, and PCR inhibitors containing poor-quality samples

Standard	Concentration (IU/µl)	Desired Ct
		HBV	IC
		(FAM/ Green)	(HEX/VIC/Yellow)
HBV Standard 1	5 X 104	17 ± 2	24 ± 3
HBV Standard 2	5 X 103	20 ± 2	24 ± 3
HBV Standard 3	5 X 102	23 ± 2	24 ± 3
HBV Standard 4	5 X 101	27 ± 2	24 ± 3
HBV Standard 5	5 X 100	31 ± 2	24 ± 3

Table 7: HBV standards and its concentration of the HBV target and desired Ct values

Sample	Expected Ct	Obtained Ct for HBV			Average Ct	Standard Deviation	CV %
		Replicate 1	Replicate 2	Replicate 3
HBV Standard 1	17 ± 2	17.01	16.89	17.07	16.99	0.09	0.54
HBV Standard 2	20 ± 2	20.29	20.32	20.34	20.32	0.03	0.12
HBV Standard 3	23 ± 2	23.61	23.88	23.73	23.74	0.14	0.57
HBV Standard 4	27 ± 2	26.95	27.03	27.12	27.03	0.09	0.31
HBV Standard 5	31 ± 2	30.58	30.91	31	30.83	0.22	0.72

Table 8: Obtained Ct values, average, standard deviation, and coefficient of variation (CV%) of 5 HBV Standards.

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 the kit is slightly lower than the reagent (NIBSC code 10/266) supplier's assigned value, still the data is satisfactory as the log10 difference between the two values is only 0.157 which is less than the cutoff value (0.5)²².

The viral load kit used in this protocol is highly robust and gives reproducible data, as shown in Figure 7. In this experiment, all 5 standards and NTC were run in triplicate, and their Ct values, average, standard deviation, and coefficient of variation (CV%) are shown in Table 8. A lower CV% of less than 1%, as mentioned in Table 8, indicates that the experiment is more reliable and produces highly accurate results.

A very low load reference sample (NIBSC code: 14/198)²³ was used with the kit to check its LoD. NIBSC code 14/198 contains less than 50 IU/mL of HBV DNA per the supplier information. DNA was extracted from NIBSC code 14/198, and multiple dilutions (1.25 IU/mL, 2.5 IU/mL, 5 IU/mL, and 25 IU/mL) of the DNA were prepared. The kit used all the dilutions and 5 HBV standards as template DNA. It was observed that that kit can detect all dilutions up to 2.5 IU/mL of HBV DNA (Figure 8), and it exactly matches the LoD of the viral load kit. Hence, the viral load kit is a highly sensitive and reproducible tool that molecular diagnostic laboratories can use to aid healthcare professionals in diagnosing and managing HBV infections effectively.

Users should keep in mind the following critical steps when they perform the procedure. The users must employ only high-quality extracted DNA as the PCR template to ensure accurate results. It is important to avoid multiple freeze-thaw cycles (not more than 3 times) of the reagents. The PCR mix should not be exposed to light for a long time as it contains the light-sensitive fluorescent probe and passive reference dye ROX. It is critical to always include no template control and standards in each run to ensure the accuracy and reliability of the results. Calibrated pipettes with sterile filtered tips must be used always. The template DNA and standards must be added in a separate area with a different pipette from the reaction preparation area to avoid cross-contamination.

The following are some common troubleshooting tips for real-time PCR-based detection and quantitation of hepatitis B virus DNA. Amplification signal in negative control may happen due to cross-contamination during reaction setup. So, the user should check for contamination of kit components. No amplification signal with standards may occur due to the following reasons: (i) Incorrect PCR mixture used. The user should check whether all components are added correctly or not. (ii) Use of expired reagents. The user must check the expiry date before the reaction is set up. (iii) Improper storage of reagents. Ensure that the reagents are stored at -20 °C or below and not kept at room temperature for a long time during reaction setup. (iv) Wrong PCR cycling condition was used. In such a case, it is recommended to repeat the PCR with the correct program. (iv) Weak or no signal of the internal control for samples may occur due to high HBV load in the sample and PCR inhibition. The user could dilute the DNA sample (1:10) and repeat the assay for high HBV load in the sample. In the case of PCR inhibition, the sample DNA must be re-extracted with good-quality reagents, and the assay must be repeated.

Real-time PCR-based detection and quantitation of hepatitis B virus DNA is a very sensitive and specific technique, but it does have some limitations like cost and complexity. Real-time PCR instruments and reagents can be expensive, and the cost of testing can be a barrier for some patients and healthcare systems. Real-time PCR is a complex technique requiring trained personnel to perform the assay and interpret the results. It is an indirect measurement of the viral load, so both the quality of the standards and template DNA may influence the result. Novel genotype of the HBV and/or mutation(s) in the primer-probe region may lead to false negative results.

Real-time PCR-based detection and quantification of HBV DNA is a highly sensitive and specific method that can detect low levels of viral DNA. In comparison to other methods like serological tests (ELISA and lateral flow assay), real-time PCR has several advantages. Firstly, it is more sensitive than the serological tests. Secondly, it can quantify the amount of HBV DNA present in a sample, whereas serological tests can only provide a qualitative result. Finally, real-time PCR is less prone to false positives than the serological tests²⁴. Overall, real-time PCR is the most sensitive, specific, and quantitative method available for the detection and quantitation of HBV DNA. It is also a relatively fast technique, which makes it ideal for clinical use.

Real-time PCR-based detection and quantitation of hepatitis B virus DNA is a powerful tool with several future applications, including developing new antiviral therapies and point-of-care testing. The technique can be used to screen new antiviral drugs and to monitor the efficacy of these drugs in clinical trials. This could lead to the development of more effective and less toxic treatments for HBV infection. Real-time PCR instruments are becoming smaller and more portable, making it possible to perform HBV viral load testing at the point of care. This could improve access to testing and lead to earlier diagnosis and treatment of HBV infection. The regents of this real-time PCR-based kit can be adapted with digital PCR for absolute quantification for more accurate monitoring of the vial load.

In addition to these specific applications, real-time PCR-based detection and quantitation of HBV DNA is likely to play an important role in the research and development of new tools and strategies for preventing, diagnosing, and treating HBV infection.

Materials

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