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

Satyabrata Bag; Santanu Ghosh; Roopa Lalwani; Pratishtha Vishnoi; Sudheer Gupta; Dhirendra Dubey

doi:10.3791/66249

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

JoVE Journal
Biology

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JoVE Journal Biology

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

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04:11 min

December 15, 2023

DOI: 10.3791/66249-v

Satyabrata Bag¹, Santanu Ghosh¹, Roopa Lalwani¹, Pratishtha Vishnoi¹, Sudheer Gupta¹, Dhirendra Dubey¹

¹3B BlackBio Biotech India Limited

Overview

This study presents a real-time PCR-based protocol for the detection and quantification of hepatitis B virus (HBV) DNA, which is essential for accurate diagnosis and monitoring of HBV infections. The method is recognized as the gold standard due to its sensitivity and specificity in identifying HBV viral loads.

Key Study Components

Research Area

Hepatitis B virus (HBV) detection
Viral load quantification
Clinical diagnostics

Background

Importance of accurate HBV detection in clinical settings
Challenges in identifying low viral loads and occult infections
Comparison of PCR with other diagnostic techniques

Methods Used

Real-time polymerase chain reaction (PCR)
Samples include HBV positive controls
Use of WHO international standard for calibration

Main Results

Successful quantification of HBV DNA using a standard curve
Identification of samples with significant amplification
Parameter calibration ensuring high accuracy and reproducibility

Conclusions

This study provides a reliable protocol for HBV DNA detection and viral load measurement.
The method’s high sensitivity makes it a valuable tool in hepatitis research and clinical diagnostics.

Frequently Asked Questions

What is the significance of detecting HBV DNA?

Detecting HBV DNA is crucial for diagnosing HBV infections and monitoring treatment effectiveness.

How does real-time PCR work?

Real-time PCR amplifies DNA and simultaneously quantifies it using fluorescent dyes during the PCR cycle.

What challenges exist in HBV detection?

Challenges include detecting low viral loads and accommodating various HBV genotypes.

How is the accuracy of PCR results ensured?

Accuracy is ensured by standardizing threshold settings across samples and validating with controls.

What role do quantification standards play?

Quantification standards provide a reference for measuring unknown samples, ensuring reliable results.

Can this protocol detect new mutations?

While the protocol is sensitive, emerging mutations may still pose challenges in accurate detection.

Is this method suitable for clinical use?

Yes, this real-time PCR method is fast, sensitive, and suited for clinical diagnostics.

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.

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 based true PCR HBV viral load kit. HBV diagnosis research uses realtime 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.

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