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Hepatitis B: A Comprehensive Guide to Real-time PCR for Diagnosing Hepatitis B

Updated: Oct 18, 2023

Introduction to Hepatitis B:

Hepatitis B is a serious liver infection caused by the hepatitis B virus (HBV). Hepatitis B is spread when blood, semen, or other body fluids from a person infected with the virus enter the body of someone who is not infected. This can happen through sexual contact; sharing needles, syringes, or other drug-injection equipment; or during pregnancy or delivery.

Not all newly infected individuals with HBV show symptoms, but for those who, symptoms may include

  • Fatigue

  • Poor appetite

  • Stomach pain

  • Nausea

  • Jaundice

For many people, hepatitis B is a short-term illness that lasts a few weeks to several months

For others, it can become a long-term, chronic infection that can remain in the body for longer than 6 months and can lead to serious, even life-threatening health issues like liver disease or liver cancer.

About two-thirds of patients with acute HBV infection have a mild, asymptomatic, and subclinical illness that usually goes undetected. Approximately one-third of adults with acute HBV infection develop clinical symptoms and signs of hepatitis, which range from mild constitutional symptoms of fatigue and nausea to more marked symptoms and jaundice, and rarely to acute liver failure.

The clinical incubation period of acute hepatitis B averages 2–3 months and can range from 1–6 months after exposure, the length of the incubation period correlating, to some extent, with the level of virus exposure. Chronic hepatitis B has a variable and dynamic course. An estimated one-third of persons with chronic HBV infection will ultimately develop a long-term consequence of the disease, such as cirrhosis, end-stage liver disease, or HCC Hepatocellular Carcinoma.

The determinants of the outcome of chronic hepatitis B appear to be both viral (HBV DNA levels, HBV genotype, some HBV mutation patterns) and host-specific (age, gender, genetic background, immune status).

Age plays a role in whether hepatitis B will become chronic. The younger a person is when infected with the hepatitis B virus, the greater the chance of developing chronic infection. About 9 in 10 infants who become infected go on to develop life-long, chronic infections. The risk goes down as a child gets older. About one in three children who get infected before age 6 will develop chronic hepatitis B.

By contrast, almost all children 6 years and older and adults infected with the hepatitis B virus recover completely and do not develop chronic infection.

The best way to prevent hepatitis B is to get vaccinated. All adults aged 18-59 should receive the vaccine and any adult who requests it may get the vaccine. All adults 18 years and older should get screened at least once in their lifetime.

Hepatitis B infection acquired in adulthood leads to chronic hepatitis in less than 5% of cases, whereas infection in infancy and early childhood leads to chronic hepatitis in about 95% of cases. This is the basis for strengthening and prioritizing infant and childhood vaccination.

World Hepatitis Day, 28 July, is an opportunity to step up national and international efforts on hepatitis, encourage actions and engagement by individuals, partners, and the public, and highlight the need for a greater global response as outlined in the WHO's Global Hepatitis Report of 2017.

Image of Dr. Baruch Blumberg
Dr. Baruch Blumberg. Image source:

The date of 28 July was chosen because it is the birthday of Nobel-prize-winning scientist Dr. Baruch Blumberg (in Image), who discovered the hepatitis B virus (HBV) and developed a diagnostic test and vaccine for the virus.

Life Cycle of the Hepatitis B Virus:

The hepatitis B virus can survive outside the body for at least 7 days. During this time, the virus can still cause infection if it enters the body of an unvaccinated individual. The incubation period of the hepatitis B virus ranges from 30 to 180 days & virus may be detected within 30 to 60 days after infection. Persistent infection can develop into chronic hepatitis B, especially when transmitted in infancy or childhood.

The Replication of Hepatitis B Virus
The replication cycle of HBV. Source: Hepatology. Author manuscript; available in PMC 2010 Jan 20. Published in final edited form as: Hepatology. 2009 May; 49(5 Suppl): S13–S21. doi: 10.1002/hep.22881


The hepatitis B virus (HBV) is a small DNA virus with unusual features similar to retroviruses. It is a prototype virus of the Hepadnaviridae family. Based on sequence comparison, HBV is classified into eight genotypes, A to H. Each genotype has a distinct geographic distribution.

The infectious HBV virion (Dane particle) has a spherical, double-shelled structure 42 nm in diameter, consisting of a lipid envelope containing HBsAg surrounding an inner nucleocapsid composed of hepatitis B core antigen (HBcAg) complexed with virally encoded polymerase and the viral DNA genome.

The genome of HBV is a partially double-stranded circular DNA of about 3.2 kilobases (kb) pairs. The viral polymerase is covalently attached to the 5′ end of the minus strand. The viral genome encodes four overlapping open reading frames (ORFs: S, C, P, and X).

The S ORF encodes the viral surface envelope proteins, the HBsAg, and can be structurally and functionally divided into the pre-S1, pre-S2, and S regions. The core of the C gene has pre-core and core regions. Multiple in-frame translation initiation codons are a feature of the S and C genes, which give rise to related but functionally distinct proteins. The C ORF encodes either the viral nucleocapsid HBcAg or hepatitis B e antigen (HBeAg) depending on whether the translation is initiated from the core or pre-core regions, respectively.

Hepatology. Author manuscript; available in PMC 2010 Jan 20. Published in final edited form as: Hepatology. 2009 May; 49(5 Suppl): S13–S21. doi: 10.1002/hep.22881
Electron micrograph of circulating forms of HBV particles in the blood is shown at the top and a schematic drawing of Dane particle, the infectious HBV particle, is shown at the bottom with various structural features.

Hepatology. Author manuscript; available in PMC 2010 Jan 20. Published in final edited form as: Hepatology. 2009 May; 49(5 Suppl): S13–S21. doi: 10.1002/hep.22881
The HBV genome. (A) The genomic organization, RNA transcripts and gene products are shown with several key regulatory elements. (B) The transcription start sites of various HBV transcripts and the proteins they encode .


During 2020, 44 states reported 2,157 acute hepatitis B cases resulting in an estimated 14,000 infections. After a decade of stable rates, the rate of acute hepatitis B abruptly decreased by 32% in 2019. This decrease may be related to fewer people seeking healthcare and being tested for hepatitis B during the COVID-19 pandemic. The number of reported acute hepatitis B cases decreased by 32% from 2019 through 2020 76% of all acute hepatitis B cases were persons aged 30-59 years.

During 2020, a total of 11,635 newly identified cases of chronic hepatitis B were reported to the CDC, corresponding to a rate of 5.0 cases per 100,000 people. The rate of newly reported chronic hepatitis B cases among Asian/Pacific Islander persons (17.6 cases per 100,000 people) was almost 12 times the rate among non-Hispanic White persons (1.5 cases per 100,000 people).

Recent News:

Hepatitis B and C infections that could be precursors to cancer of the liver at a later stage have become notifiable diseases in Bengal. The state health department has shared a format, through which each healthcare unit and diagnostic center in the state has to notify it as and when they get cases of the two viral infections.

Health experts termed this an important step for early detection, prevention, and medical intervention of the affected. According to health officials, the prevalence of hepatitis B in Bengal is 1.3 percent against a national average of 1 percent while the prevalence of hepatitis C is 0.3 percent against a national average of 0.7 percent.

But health experts say this estimate could be only the tip of the iceberg as in most cases, the infection remains. "Both hepatitis B and C can be a precursor to cancer of the liver. While B can be kept under control, C is curable. Therefore, early detection, and assessing the treatable, and timely intervention can make a lot of difference."

In 2021 when the pandemic was raging, the state government set up a network of three high-end state referral labs to combat hepatitis B and C. While the apex lab is situated at IPGMER, Kolkata, the other two are at Burdwan Medical College and North Bengal Medical College.

Hepatitis B and C are most visible when the disease becomes acute or chronic.


WHO recommends that all blood donations be tested for hepatitis B to ensure blood safety and avoid accidental transmission.

WHO recommends that all adults have access to and be offered HBsAg testing with linkage to prevention, care, and treatment services as needed.

The diagnosis of acute hepatitis B is reliably made by the finding of IgM anti-HBc in serum, particularly in a patient with HBsAg and signs, symptoms, or laboratory features of acute hepatitis. Nevertheless, in some instances, HBsAg is cleared rapidly from the serum, and IgM anti-HBc is the only marker detectable when the patient presents with hepatitis.

Testing for anti-HBc (total) and anti-HBs is not useful in diagnosis, and testing for HBeAg and anti-HBe should be reserved for persons who test positive for HBsAg. The finding of HBsAg without IgM anti-HBc suggests the presence of chronic hepatitis B, but this diagnosis generally also rests upon a finding of the persistence of HBsAg for at least 6 months.

HBV DNA testing can also be helpful in the assessment of the level of viral replication and possibly helpful in assessing prognosis and the need for antiviral therapy. Assays for HBV DNA levels have improved substantially over the years.

The current real-time polymerase chain reaction–based assay (TaqMan) has a lower limit of detection of 5–10 HBV DNA copies/mL and can accurately quantify a wide range of levels. With this degree of sensitivity, HBV DNA can be detected early during infection, arising before the appearance of other serological markers, such as HBsAg or anti-HBc.

As a consequence, testing for HBV DNA has emerged as a primary approach in the diagnosis and management of HBV infection. HBV DNA testing has now become routinely used in blood product screening (nucleic acid testing) and monitoring of patients with HBV during treatment.

Persistently high levels of HBV DNA following the resolution of hepatitis may be indicative of a failure to control the infection and evolution into a chronic infection.


There is no specific treatment for acute hepatitis B. Therefore, care is aimed at maintaining comfort and adequate nutritional balance, including the replacement of fluids lost from vomiting and diarrhea. Most important is the avoidance of unnecessary medications. Acetaminophen, paracetamol, and medication against vomiting should be avoided.

Chronic hepatitis B infection can be treated with medicines, including oral antiviral agents. Treatment can slow the progression of cirrhosis, reduce the incidence of liver cancer, and improve long-term survival. In 2021 WHO estimated that 12% to 25% of people with chronic hepatitis B infection will require treatment, depending on setting and eligibility criteria.

WHO recommends the use of oral treatments (tenofovir or entecavir) as the most potent drugs to suppress the hepatitis B virus. Most people who start hepatitis B treatment must continue it for life.

In low-income settings, most people with liver cancer die within months of diagnosis. In high-income countries, patients present to hospitals earlier in the course of the disease, and have access to surgery and chemotherapy which can prolong life for several months to a few years. Liver transplantation is sometimes used in people with cirrhosis or liver cancer in high-income countries, with varying success.


WHO recommends that all infants receive the hepatitis B vaccine as soon as possible after birth, preferably within 24 hours, followed by 2 or 3 doses of the hepatitis B vaccine at least 4 weeks apart to complete the vaccination series. Protection lasts at least 20 years and is probably lifelong.

WHO does not recommend booster vaccinations for persons who have completed the 3-dose vaccination schedule.

In addition to infant vaccination, WHO recommends the use of antiviral prophylaxis for the prevention of hepatitis B transmission from mother to child. Implementation of blood safety strategies and safer sex practices, including minimizing the number of partners and using barrier protective measures (condoms), also protect against transmission.

WHO response:

In May 2016, the World Health Assembly adopted the first Global health sector strategy on viral hepatitis, 2016–2020. The strategy highlighted the critical role of universal health coverage and set targets that align with those of the Sustainable Development Goals.

The strategy proposed the elimination of viral hepatitis as a public health threat by 2030 (defined as a 90% reduction in new chronic infections and a 65% reduction in mortality, compared with the 2015 baseline), and included a roadmap towards elimination by implementing key prevention, diagnosis, treatment and community interventions strategies.

In May 2022 the 75th World Health Assembly noted a new set of integrated global health sector strategies on HIV, viral hepatitis, and sexually transmitted infections for the period of 2022–2030. Based on these previous and now new strategies, a broad range of Member States have developed comprehensive national hepatitis programs and elimination strategies guided by the global health sector strategy.

To support countries in achieving the global hepatitis elimination targets under the Sustainable Development Agenda 2030, WHO is working to:

  • Raise awareness, promote partnerships, and mobilize resources

  • Formulate evidence-based policy and data for action

  • Increase health equities within the hepatitis response

  • Prevent transmission

  • Scale up screening, care, and treatment services.

WHO organizes the annual World Hepatitis Day campaign (as 1 of its 9 flagship annual health campaigns) to increase awareness and understanding of viral hepatitis. For World Hepatitis Day 2022, WHO focuses on the theme “Bringing hepatitis care closer to you” and calls for simplified service delivery of viral hepatitis services, bringing care closer to communities.

NeoDx HBV Qualitative RT-PCR Detection Kit:

It can detect HBV infection from the initial stage to the final stage of the infection. The test will confirm the presence or absence of the HBV infection. After the confirmation of the infection, a quantitative test is recommended for the quantification of the HBV viral DNA in the patient.

NeoDx HBV Quantitative RT-PCR Detection Kit:

It can measure the quantity of the HBV viral gene in the blood, plasma, or serum. The quantitative standards with the known copy numbers should be calibrated against the WHO International Standard 4th WHO International Standard for HBV DNA for NAT NIBSC code: 10/266 for the quantitation of HBV viral DNA for assigning copy numbers to a measured level of HBV DNA.

Salient Features and Benefits of NeoDx HBV Qualitative RT-PCR Detection Kit and HBV Quantitative RT-PCR Detection Kit

  • Easy calculations to determine the Copy number and IU/mL which makes the reporting more accurate and convenient.

  • Multiplex, single tube assay involves amplification of HBV DNA with the internal control gene.

  • Minimal pipetting steps during the preparation of the master mix.

  • In-silico analysis was performed for all the primer sequences and probe sequences used in the HBV Qualitative RT PCR Detection Kit primer and probe sets are specific to the Hepatitis B viral gene and human internal control respectively.

  • All the primers and probes sequences' specificity and efficiency were analyzed and have 100% specificity in identifying the respective sequences.

  • NIBSC calibrated standards for the quantification of the Hepatitis B viral DNA. (4th WHO International Standard for HBV DNA (NIBSC code: 10/266))

  • High-Quality Mastermix which shows Increased Inhibitor Tolerance, Thermostability, and Reproducibility.

  • The quantitative kit can detect the target gene up to 32 IU/ML.

  • Open instrument compatibility.

  • Fast RT-PCR Run time.

HBV Qualitative RT-PCR Detection Kit

  • HBV Qualitative RT-PCR Detection Kit is an in-vitro RT-PCR qualitative assay for the specific detection of HBV in human plasma, serum, and whole blood.

  • HBV Qualitative RT-PCR is a Multiplex assay kit where the detection of 2 genes (HBV gene and one human internal control) can be detected in a single-tube reaction.

  • The test will confirm the presence or absence of the HBV infection.

  • Fast RT-PCR Run time.

  • Compatible with all RT-PCR instruments

HBV Quantitative RT-PCR Detection Kit

  • HBV Quantitative RT-PCR Detection Kit is an in-vitro RT-PCR quantitative assay for the specific detection of HBV in human plasma, serum, and whole blood.

  • HBV Quantitative RT-PCR is a Multiplex assay kit where the detection of 2 genes (HBV gene and one human internal control) can be detected in a single-tube reaction.

  • Easy calculations to determine the Copy number and IU/mL which makes the reporting more accurate and convenient.

  • NIBSC calibrated standards for the quantification of the HBV DNA. (4th WHO International Standard for HBV DNA (NIBSC code: 10/266)).

  • Fast RT-PCR Run time.

  • Compatible with all RT-PCR instruments.

Applications of NeoDx HBV Qualitative and Quantitative RT-PCR Detection Kits.

  • Highly specific and sensitive detect, and quantify the HBV viral infection.

  • To identify the extent of infection whether it is acute or chronic infection.

  • To screen the Blood and organ donor and recipient for HBV infection before blood transfusion and organ transplantation.

  • Avoid transmission from the Mother to the baby by early diagnosis.

  • To evaluate the treatment response by monitoring viral copy numbers.

  • To check the effectiveness of the vaccine against HBV.

Difference between the commercially available RT-PCR kits for HBV and NeoDx HBV RT-PCR detection kits.

  • Prediluted, ready-to-use five Quantitative Standards (QS1- QS5).

  • No need for additional internal control to be added before DNA extraction.

  • Fast PCR run time ( approx 80 mins)

  • High-quality Mastermix which shows Increased Inhibitor Tolerance, Thermostability, and Reproducibility.

NeoDx Biotech Labs provides the most reliable qPCR kits to HBV patients and helps them with successful treatment. The highly specific and sensitive kits detect, and quantify the HBV viral infection.

Order Information:

ProductCat#/SKU/Pack Size

  1. HBV Qualitative RT-PCR Detection Kit NDXIVD013_02: 20 Rxns NDXIVD013_01: 50 Rxns NDXIVD013: 100 Rxns

  2. HBV Quantitative RT-PCR Detection kit NDXIVD008_02: 20 Rxns NDXIVD008_01: 50 Rxns NDXIVD008: 100 Rxns

Sample Kits Available

Developed and Manufactured by

NeoDx Biotech Labs Pvt Ltd

SR4, 4th floor, Pragathi Plaza, Opp. Biocon, Hebbagodi, Bengaluru, Karnataka India - 560100


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