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

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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.

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Data availability

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

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Funding

This study was supported by Istanbul Gelisim University Office of Scientific Research Projects, Grant No.: KAP-050421-AHH.

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Authors and Affiliations

  1. Life Sciences and Biomedical Engineering Application and Research Centre, Istanbul Gelisim University, Istanbul, Türkiye

    Abbas Ali Husseini & Serap Yesilkir Baydar

  2. Vocational School of health services, Istanbul Gelisim University, 34310, Istanbul, Türkiye

    Abbas Ali Husseini

  3. Department of Biomedical Engineering, Faculty of Engineering and Architecture, Istanbul Gelisim University, Istanbul, Türkiye

    Serap Yesilkir Baydar

Authors
  1. Abbas Ali Husseini
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Contributions

AAH conceived the study, planned and performed the experiments, analyzed the data, and supervised the project. AHH and SYB drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Abbas Ali Husseini.

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Conflict of interest

there are no conflicts of interest to declare.

Ethical approval

All participants were provided with informed consent and the study protocol has been approved by the Ethics Committee of the Faculty of medicine, Marmara University in Istanbul, Türkiye (07.05.2021 date & protocol no: 09.2021.577).

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Husseini, A.A., Baydar, S.Y. Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection. Mol Biol Rep 50, 8329–8336 (2023). https://doi.org/10.1007/s11033-023-08730-9

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  • DOI: https://doi.org/10.1007/s11033-023-08730-9

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