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. Store the extracted DNA at -20 &#176;C until further use in PCR. 
	&#8203;NOTE: The recommended volume of serum or plasma for DNA extraction is 500 &#181;L, and the elution volume is 50 &#181;L. In this study, the viral DNA was extracted using a viral nucleic acid extraction kit (Table of Materials).</li>
</ol>
2. Real-time PCR
<ol>
	<li>Thaw all the reagents (Table 1) of the real-time PCR kit at room temperature (RT; 15-25 &#176;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 &#215; g for 15 s at RT. Keep all the thawed components on a cooling block.</li>
	<li>Reaction preparation
	<ol>
		<li>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).</li>
		<li>Pipet 15 &#181;L of the above-prepared PCR mix into each PCR tube. Then, add 15 &#181;L of the extracted sample DNA. Add 15 &#181;L of at least one of the standards for HBV (HBV Standard 1-5) as a positive control (PC) for detecting HBV DNA and 15 &#181;L 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.</li>
		<li>Close the tubes, mix the components by pulse vortexing for 10 s at a moderate speed, and centrifuge at 8,000 &#215; 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.</li>
	</ol>
	</li>
	<li>Program setup
	<ol>
		<li>Program the thermal cycler using the cycling conditions as recommended in Table 3.</li>
	</ol>
	</li>
	<li>Channel/Fluorophore Selection
	<ol>
		<li>Select the fluorescent dyes for commonly used real-time PCR platforms, as mentioned in Table 4.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>After the run is completed, analyze the amplification plot on a linear scale.</li>
		<li>Threshold setting
		<ol>
			<li>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. 
			&#8203;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.</li>
		</ol>
		</li>
		<li>Cutoff
		<ol>
			<li>Run the kit for 40 amplification cycles. Do not consider any amplification beyond 38 cycles for any interpretation.</li>
		</ol>
		</li>
		<li>Qualitative result analysis
		<ol>
			<li>Use the kit for qualitative detection of HBV DNA. Use Standard 2 as PC with expected Ct values at 20 &#177; 2 for HBV and 24 &#177; 3 for IC.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Quantitative result analysis
		<ol>
			<li>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).</li>
			<li>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/&#181;L).</li>
			<li>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.</li>
			<li>The software will calculate the concentration of each HBV-positive sample in international units per microliter (IU/&#181;L). To calculate the viral load (conversion of IU/&#181;L to international units per milliliter [IU/mL]), use the following equation: 
			Result (IU/mL) = (Result [IU/&#181;L] x Elution Volume [&#181;L])/ Sample Volume (mL) 
			NOTE: The 4th 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 &#181;L 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/&#181;L x 50 &#181;L)/0.5 mL = 6,65,900 IU/mL.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Detection and Quantification of Hepatitis B Virus DNA in Human Serum

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	<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><tbody><tr><td>4&lt;sup&gt;th&lt;/sup&gt; 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&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>ABI 7500; QuantStudio 5</td><td>This is a real time PCR machine&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>TRUPCR HBV Viral Load kit&amp;nbsp;</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.

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

Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection 

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.

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 ...

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

Research

JoVE Journal

Biology

3.1K Views.  3B BlackBio Biotech India Limited. In deze video laten we u een stapsgewijs protocol zien om HBV-DNA te detecteren en de virale lading ervan te kwantificeren met behulp van onze IVD-goedgekeurde real-time PCR-afval, via PCR HBV-virale ladingskit. HBV-diagnoseonderzoek maakt gebruik van real-time PCR, digitale PCR, NGS en serologische tests. Realtime PCR is de gouden standaard voor HBV-DNA-detectie en kwantificering van virale lading.Digitale PCR kwantificeert de absolute virale lading. NGS identificeert nieuwe mutaties ...

Video: Auteur in de schijnwerpers: vooruitgang en uitdagingen bij de detectie van hepatitis B-virussen 

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

Immunology and Infection

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection

Plate HepG2 cells, stably expressing sodium taurocholate, co-transporting polypeptide, or NTCP that are susceptible to HBV infection, and incubate at 37 degrees Celsius and 5% CO2.
One day before infection, plate approximately 5 x 104 HepG2/NTCP cells into the wells of a 96-well collagen-coated plate in 0.1 milliliters of culture medium. Thaw recombinant HPV in a 37 degrees Celsius water bath, until there is a small bit of ice remaining in the vial.
Prepare medium for infection by combining 78 microliters of fresh culture medium, two microliters of DMSO, 10 microliters of 40% PEG8000 in 1X PBS, and 10 microliters of recombinant HPV. Add 100 microliters of recombinant HBV solution to each well of a 96-well plate.
One day after infection, use 300 microliters of PBS to wash the cell three times, to remove the contaminating reporter protein from the virus fraction. Add 200 microliters of culture medium containing 2% DMSO to the infected cells, and incubate for one week or less.
To carry out analysis of the HBV infection, one week after infection, use PBS to wash the infected cells three times. Add 50 microliters of lysis buffer to the infected cells. Rock the culture plate for five minutes, then, centrifuge at 2000 times g for five minutes. Add 50 microliters of the reporter substrate to a luminometer plate, then, add 20 microliters of the cell lysate to the plate, and mix by briefly shaking. Finally, place the plate in the luminometer and initiate reading.

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

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