A multi-template multiplex PCR assay for hepatitis B virus and human β-globin

Area of Science:

• Molecular Biology
• Virology
• Infectious Diseases

Background:

• Hepatitis B virus (HBV) infection diagnosis relies on serological markers like Hepatitis B surface antigen (HBsAg) and antibodies.
• Molecular detection of HBV DNA is crucial, especially in clinical settings, and requires reliable internal controls for sample quality assessment.
• Human β-globin is a common housekeeping gene used as an internal control in molecular diagnostics.

Purpose of the Study:

• To construct plasmid clones of human β-globin and HBs genes for assay development.
• To characterize a multiplex PCR assay for simultaneous detection of HBsAg DNA and β-globin.
• To evaluate the sensitivity and potential of the multiplex assay as a diagnostic tool.

Main Methods:

• Construction of individual plasmid clones for human β-globin and HBs genes.
• Development and characterization of a multiplex PCR assay.
• Assessment of detection limits using conventional and multiplex PCR conditions.
• Evaluation of co-amplification effects on assay sensitivity.

Main Results:

• The developed assay demonstrated detection limits below 10 genome copies of HBsAg DNA and β-globin in vitro.
• Co-amplification of HBsAg DNA and β-globin under multiplex conditions impacted assay sensitivity.
• The study established the feasibility of using plasmid constructs for assay characterization.

Conclusions:

• The multiplex PCR assay shows promise for sensitive molecular diagnosis of HBV infection.
• This assay could serve as a valuable complement to traditional serological methods, particularly in resource-limited settings.
• Further optimization is needed to fully realize the diagnostic potential of this molecular approach.