Simultaneous detection of precore/basal core promoter mutations in hepatitis B virus using arrayed primer extension

Area of Science:

• Hepatology
• Molecular Virology
• Diagnostic Technologies

Background:

• Hepatitis B virus (HBV) infection poses a significant global health burden.
• Precore/basal core promoter mutations in HBV are linked to severe liver conditions like cirrhosis and cancer.
• Simultaneous detection of these mutations is crucial for managing HBV-infected patients.

Purpose of the Study:

• To develop and apply arrayed primer extension (APEX) for detecting single nucleotide polymorphisms (SNPs) in the HBV precore/basal core promoter region.
• To enable simultaneous screening of multiple HBV mutations.

Main Methods:

• Optimization of the APEX assay for simultaneous detection of eight potential HBV SNP sites.
• PCR amplification of HBV precore/basal core promoter regions from 36 patients.
• Hybridization of purified, fragmented HBV DNA with immobilized oligonucleotides, followed by fluorescently labeled dideoxynucleotide incorporation.

Main Results:

• The optimized APEX method achieved simultaneous detection of multiple HBV SNPs with high efficiency, sensitivity, and specificity.
• APEX successfully discriminated between wild-type and mutant HBV genotypes.
• The assay detected mixed genotypes, which were missed by conventional DNA sequencing.

Conclusions:

• APEX provides a rapid and accurate method for simultaneous SNP detection in the HBV precore/basal core promoter.
• This technique facilitates large-scale diagnostic analysis for HBV.
• The APEX approach is adaptable for broader screening across the entire HBV genome.