We acknowledge Praveen, Kusum, Chandan, Isha, Rashmi, Babli, Shivani, Ankita, and Shikha for their technical assistance.

This study did not involve any human participants or clinical samples. The only biological material used was the 4th 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
<ol>
	<li>Extract the viral DNA from human serum or plasma sample. ...

Detection and Quantification of Hepatitis B Virus DNA in Human Serum

<ol>
	<li>Ryu, W. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Virology of Human Pathogenic Viruses. , (2016).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hepatitis+b+virus+infection+in+the+Asia-Pacific+region+and+Africa:+Review+of+disease+progression.">Chronic hepatitis b virus infection in the Asia-Pacific region and Africa: Review of disease progression.</a> Journal of Gastroenterology and Hepatology. 20 (6), 833-843 (2005).</li><li>Iloeje, U. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+cirrhosis+risk+based+on+the+level+of+circulating+Hepatitis+B+viral+load.">Predicting cirrhosis risk based on the level of circulating Hepatitis B viral load.</a> Gastroenterology. 130 (3), 678-686 (2006).</li><li>Huang, Y. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+risk+and+sex+difference+of+hepatocellular+carcinoma+among+patients+with+chronic+Hepatitis+B+and+C.">Lifetime risk and sex difference of hepatocellular carcinoma among patients with chronic Hepatitis B and C.</a> Journal of Clinical Oncology. 29 (27), 3643 (2011).</li><li>. World Health Organization fact sheet Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets">https://www.who.int/news-room/fact-sheets</a> (2023)</li><li>Watanabe, M., Toyoda, H., Kawabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+assay+of+hepatitis+b+virus+DNA+in+human+serum+and+plasma+by+fully+automated+genetic+analyzer+mutaswako+g1.">Rapid quantification assay of hepatitis b virus DNA in human serum and plasma by fully automated genetic analyzer mutaswako g1.</a> PLoS One. 18 (2), e0278143 (2023).</li><li>Chan, H. L. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+genotype+and+Hepatitis+B+virus+DNA+levels+are+correlated+with+histological+liver+damage+in+HBeAg-negative+chronic+Hepatitis+B+virus+infection.">Viral genotype and Hepatitis B virus DNA levels are correlated with histological liver damage in HBeAg-negative chronic Hepatitis B virus infection.</a> The American Journal of Gastroenterology. 97 (2), 406-412 (2002).</li><li>Liu, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+factors+correlate+with+Hepatitis+B+e+antigen+seroconverson+in+patients+with+chronic+Hepatitis+B.">Viral factors correlate with Hepatitis B e antigen seroconverson in patients with chronic Hepatitis B.</a> Liver International. 26 (8), 949-955 (2006).</li><li>Liu, C. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Hepatitis+B+viral+load+and+basal+core+promoter+mutation+in+hepatocellular+carcinoma+in+Hepatitis+B+carriers.">Role of Hepatitis B viral load and basal core promoter mutation in hepatocellular carcinoma in Hepatitis B carriers.</a> The Journal of Infectious Diseases. 193 (9), 1258-1265 (2006).</li><li>Yeo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+viral+load+predicts+survival+of+HCC+patients+undergoing+systemic+chemotherapy.">Hepatitis B viral load predicts survival of HCC patients undergoing systemic chemotherapy.</a> Hepatology. 45 (6), 1382-1389 (2007).</li><li>Yim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levels+of+Hepatitis+B+virus+(HBV)+replication+during+the+nonreplicative+phase:+HBV+quantification+by+real-time+PCR+in+korea.">Levels of Hepatitis B virus (HBV) replication during the nonreplicative phase: HBV quantification by real-time PCR in korea.</a> Digestive Diseases and Sciences. 52, 2403-2409 (2007).</li><li>Noh, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+impact+of+viral+load+on+the+development+of+hepatocellular+carcinoma+and+liver-related+mortality+in+patients+with+hepatitis+c+virus+infection.">Clinical impact of viral load on the development of hepatocellular carcinoma and liver-related mortality in patients with hepatitis c virus infection.</a> Gastroenterology Research and Practice. 2016, 7476231 (2016).</li><li>Mendy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+viral+load+and+risk+for+liver+cirrhosis+and+hepatocellular+carcinoma+in+the+Gambia,+West+Africa.">Hepatitis B viral load and risk for liver cirrhosis and hepatocellular carcinoma in the Gambia, West Africa.</a> Journal of Viral Hepatitis. 17 (2), 115-122 (2010).</li><li>Abe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+Hepatitis+B+virus+genomic+DNA+by+real-time+detection+PCR.">Quantitation of Hepatitis B virus genomic DNA by real-time detection PCR.</a> Journal of Clinical Microbiology. 37 (9), 2899-2903 (1999).</li><li>Pas, S. D., Fries, E., De Man, R. A., Osterhaus, A. D., Niesters, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+real-time+detection+assay+for+Hepatitis+B+virus+DNA+and+comparison+with+two+commercial+assays.">Development of a quantitative real-time detection assay for Hepatitis B virus DNA and comparison with two commercial assays.</a> Journal of Clinical Microbiology. 38 (8), 2897-2901 (2000).</li><li>Ronsin, C., Pillet, A., Bali, C., Denoyel, G. R. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+cobas+ampliprep-total+nucleic+acid+isolation-cobas+Taqman+Hepatitis+B+Virus+(HBV)+quantitative+test+and+comparison+to+the+versant+HBV+DNA+3.0+assay.">Evaluation of the cobas ampliprep-total nucleic acid isolation-cobas Taqman Hepatitis B Virus (HBV) quantitative test and comparison to the versant HBV DNA 3.0 assay.</a> Journal of Clinical Microbiology. 44 (4), 1390-1399 (2006).</li><li>Sum, S. S. M., Wong, D. K. H., Yuen, J. C. H., Lai, C. L., Yuen, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+cobas+taqman&#8482;+HBV+test+with+the+cobas+amplicor+monitor&#8482;+test+for+measurement+of+Hepatitis+B+virus+DNA+in+serum.">Comparison of the cobas taqman&#8482; HBV test with the cobas amplicor monitor&#8482; test for measurement of Hepatitis B virus DNA in serum.</a> Journal of Medical Virology. 77 (4), 486-490 (2005).</li><li>Lall, S., Choudhary, M. C., Mahajan, S., Kumar, G., Gupta, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+evaluation+of+TRUPCR&#174;+HBV+real-time+PCR+assay+for+Hepatitis+B+virus+DNA+quantification+in+clinical+samples:+Report+from+a+tertiary+care+liver+centre.">Performance evaluation of TRUPCR&#174; HBV real-time PCR assay for Hepatitis B virus DNA quantification in clinical samples: Report from a tertiary care liver centre.</a> Virusdisease. 30 (2), 186-192 (2019).</li><li>Garg, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+entry+receptors+and+viral+markers+suggest+a+low+level+of+placental+replication+of+Hepatitis+B+virus+in+a+proportion+of+pregnant+women+infected+with+chronic+Hepatitis+B.">Presence of entry receptors and viral markers suggest a low level of placental replication of Hepatitis B virus in a proportion of pregnant women infected with chronic Hepatitis B.</a> Scientific Reports. 12 (1), 17795 (2022).</li><li>NIBSC-Code:10/266. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard for Hepatitis B Virus (Hbv) DNA for Nucleic Acid Amplification Techniques (NAT). , (2016).</li><li>Kim, H., Hur, M., Bae, E., Lee, K. A., Lee, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+evaluation+of+cobas+HBV+real-time+PCR+assay+on+Roche+cobas+4800+system+in+comparison+with+cobas+Ampliprep/cobas+Taqman+HBV+test.">Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 system in comparison with cobas Ampliprep/cobas Taqman HBV test.</a> Clinical Chemistry and Laboratory Medicine. 56 (7), 1133-1139 (2018).</li><li>Madihi, S., Syed, H., Lazar, F., Zyad, A., Benani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+comparison+of+the+artus+HBV+QS-RGQ+and+the+CAP/CTM+HBV+v2.0+assays+regarding+HEPATITIS+B+virus+DNA+quantification.">Performance comparison of the artus HBV QS-RGQ and the CAP/CTM HBV v2.0 assays regarding HEPATITIS B virus DNA quantification.</a> BioMed Research International. 2020, 4159189 (2020).</li><li>. Nibsc-14/198 Available from: <a target="_blank" href="https://www.nibsc.org/">https://www.nibsc.org/</a> (2023)</li><li>Guvenir, M., Arikan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus:+From+diagnosis+to+treatment.">Hepatitis B virus: From diagnosis to treatment.</a> Polish Journal of Microbiology. 69 (4), 391-399 (2020).</li></ol>

The authors are associated with Kilpest India Limited, the manufacturer of the HBV viral load kit used in this study.

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 ...

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&#39;s life, potentially leading to liver cirrhosis and hepatocellular carcinoma2,3,4. 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 success6,7,8,9,10,11. High viral load is associated with an increased risk of liver disease progression, including cirrhosis and liver cancer12,13. 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 techniques14,15,16,17. 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 used18,19, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4th WHO International Standard for hepatitis B virus DNA</td>
			<td>NIBSC</td>
			<td>NIBSC code: 10/266</td>
			<td>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).</td>
		</tr>
		<tr>
			<td>ABI real-time PCR machines&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>ABI 7500; QuantStudio 5</td>
			<td>This is a real time PCR machine&#160;</td>
		</tr>
		<tr>
			<td>HBV, HCV and HIV Multiplex 14/198</td>
			<td>NIBSC</td>
			<td>NIBSC code: 14/198</td>
			<td>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)</td>
		</tr>
		<tr>
			<td>QIAGEN real-time PCR machine</td>
			<td>Qiagen</td>
			<td>Rotor-Gene Q</td>
			<td>This is a real time PCR machine&#160;</td>
		</tr>
		<tr>
			<td>TRUPCR HBV Viral Load kit&#160;</td>
			<td>Kilpest India Limited</td>
			<td>3B294</td>
			<td>This is a real time PCR based kit used in this study for the HBV DNA detection and viral load determination</td>
		</tr>
		<tr>
			<td>TRUPCR Viral Nucleic Acid Extraction Kit</td>
			<td>Kilpest India Limited</td>
			<td>3B214</td>
			<td>This is a DNA extraction kit used for the extraction of HBV viral DNA from NIBSC:10/266 and NIBSC:14/198</td>
		</tr>
	</tbody>
</table>

real-time polymerase chain reaction-based detection and quantification of hepatitis b virus dna

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 4th 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.

Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection 

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

In this video, we will show you a step-by-step protocol to detect HBV DNA and quantify its viral load using our IVD approved real-time PCR waste, through PCR HBV viral load kit. HBV diagnosis research uses, real time PCR, digital PCR, NGS and serological tests. Realtime PCR is the gold standard for HBV DNA detection and viral load quantification.Digital PCR quantifies absolute viral load. NGS identifies new mutations and serological tests detect antigens or antibodies. There are several experimental challenges with detecting low viral load cases and occult HBV infections.Some assays may not fall from well across all HBV genotypes and emerging new mutations may lead to false negative results. Compared to other techniques the realtime PCR based protocol is the most sensitive, specific and quantitative method available for HBV detection. It is also a relatively fast technique, which makes it ideal for clinical use.

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...

Watch this Scientific Journal Video about Advancements and Challenges in Hepatitis B Virus Detection at JoVE.com

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.

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly ...

real-time-polymerase-chain-reaction-based-detection-quantification

Research

JoVE Journal

Biology

Quantifying Hepatitis B Virus (HBV) DNA in Human Serum Samples Using Real-Time PCR

To begin, thaw all reagents provided in the real-time PCR kit at room temperature. Once thawed, pulse vortex the components for 10 seconds at a moderate speed to mix them. Then, centrifuge at 8, 000G for 10 seconds at room temperature.Keep the thawed components on a cooling block. Next, prepare a PCR mix for an HBV positive sample, five HBV standards, and no template control. Thoroughly mix the added reagents by gently pipetting seven to 10 times, followed by pulse vortexing, and centrifuge at 8, 000G for 10 seconds at room temperature.Then, pipette 15 microliters of the PCR mix into each PCR tube. Add 15 microliters of extracted DNA sample, five HBV standards, and nuclease free water as a no template control. Close the PCR tubes and briefly spin in the centrifuge.Load the PCR tubes in a real-time PCR machine. Now, initiate the thermal cycler software. Choose fluorescent dyes and program the thermal cycler for real-time PCR.Post run, analyze the amplification plot using a linear scale. Set the PCR reaction threshold within the exponential phase. Maintain consistency in threshold settings for accuracy and reproducibility.Use the same threshold for all samples on a specific PCR machine. Consider samples with amplification before CD value 38 as HBV positive based on the assay's cutoff. Use five quantification standards for HBV-specific DNA, calibrated against the Fourth WHO international standard for HBV DNA.Generate a standard curve with these standards to quantify HBV DNA in unknown samples. Interpret values for unknown samples only if the standard curve slope is between minus 3.1 and minus 3.6, the R square value is between 0.99 and 1.0, PCR efficiency is within 90%to 110%and there is no amplification in the negative control.

2.0K Views.  3B BlackBio Biotech India Limited.  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.

Detection and Quantification of Hepatitis B Virus DNA in Human Serum

Summary