Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Point-of-Care Testing

Background:

• Direct amplification of nucleic acid from lysate is being explored for point-of-care (POC) diagnostics.
• Hepatitis B Virus (HBV) detection assays are crucial for disease management.

Purpose of the Study:

• To develop a rapid, precise, and cost-effective lateral flow biosensor for HBV nucleic acid detection at the point-of-care.
• To evaluate the effectiveness of direct amplification versus purified nucleic acid in Loop-mediated Isothermal Amplification coupled with Lateral Flow (LAMP-LF) biosensing.

Main Methods:

• Clinical serum samples confirmed HBV-positive by real-time PCR were used.
• Nucleic acid was prepared using spin column purification or direct heat treatment of serum.
• Loop-mediated Isothermal Amplification (LAMP) primers targeted a 250 bp fragment of the HBV polymerase gene.
• Optimal LAMP conditions were determined (67°C for 30 min).
• Detection was performed using lateral flow assay (Milenia HybriDetect1 strips).

Main Results:

• Optimized LAMP reaction at 67°C for 30 min showed significant turbidity.
• Using purified nucleic acid, the lateral flow assay detected viral loads from 10⁸ IU/ml down to 10¹ IU/ml.
• Direct amplification from heat-treated serum detected a minimum viral load of 10³ IU/ml.

Conclusions:

• The LAMP-LF assay is a promising tool for HBV testing in resource-limited settings.
• Direct amplification without DNA purification may reduce the sensitivity of the assay.