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

Abstract

Background and objective: The utilization of direct amplification of nucleic acid from lysate has attracted interest in the advancement of straightforward and economical point-of-care assays. Consequently, this study primarily focuses on the development of a rapid, precise, and cost-effective lateral flow biosensor for the convenient detection of HBV nucleic acid at the point-of-care. Furthermore, the study evaluates the effectiveness of the direct amplification method in comparison to purified nucleic acid samples within the context of LAMP-LF biosensing approaches. Methods: The experiments conducted in this study utilized clinical serum samples that were confirmed as HBV-positive through real-time PCR assays. Sample preparation involved employing spin column nucleic acid purification and serum heat treatment. To amplify a 250 bp fragment of the HBV polymerase gene, three pairs of specific LAMP primers were utilized, which were biotin-labeled and FITC-labeled for detection purposes. Various incubation temperatures (ranging from 64 to 68 °C) and durations (30 min, 45 min, and 1 h) were investigated to determine the optimal conditions for the LAMP assay. The results were subsequently assessed through fluorometric analysis, white turbidity measurements, and lateral flow assay. Milenia HybriDetect1 strips, designed for immediate use, were employed to visualize the LAMP amplicons. Furthermore, the performance of the lateral flow biosensor was evaluated using 10-fold serial dilutions of a secondary standard containing a viral load of 108 IU/ml. Results: The optimization of the LAMP reaction was achieved at a temperature of 67 °C, resulting in significant turbidity after a 30-minute incubation period. When the spin column purification method was employed, varying test bands were observed for templates ranging from 108 IU/ml to 101 IU/ml viral load. However, when serum samples underwent heat treatment and the resulting supernatant was directly used for LAMP, the lateral flow assay was capable of detecting a minimum viral load of 103 IU/ml. Conclusion: In resource-limited settings, the LAMP-LF assay presents a promising solution for HBV testing. However, it is important to note that direct amplification without DNA purification may diminish the performance of the approach.