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Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

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Abstract

Loop-mediated amplification (LAMP) is an isothermal amplification technique favored in diagnostics and point-of-care work due to its high sensitivity and ability to run in isothermal conditions. In addition, a visual readout by lateral flow strips (LFS) can be used in conjunction with LAMP, making the assay accessible at the point-of-care. However, the amplicons resulting from a LAMP reaction varied in length and shape, making them undiscernible on a double-stranded DNA intercalating dye stained gel. Standard characterization techniques also do not identify which amplicons specifically bind to the LFS, which generate the visual readout. We aimed to standardize our characterization of LAMP products during assay development by using fluorescein amidite (FAM) and biotin-tagged loop forward and backward primers during assay development. A pvuII restriction enzyme digest is applied to the LAMP products. FAM-tagged bands are directly correlated with the LFS visual readout. We applied this assay development workflow for an HPV 16 assay using both plasmid DNA and clinical samples to demonstrate proof of concept for generalized assay development work.

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Acknowledgments

The authors would like to thank the Clinical & Translation Science Institute’s Study Design and Statistical Analysis Consult group who provided additional insight on the applications of data studies on patient samples. This publication was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through BU-CTSI Grant Number 1UL1TR001430. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Funding

This work was funded by NIH U54 EB015403 and NIH U54 EB015408, both parts of the NIH Point of Care Technology Research Network (https://POCTRN.org). The Boston University Precision Diagnostics Center has funded the analysis of the patient samples

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Authors and Affiliations

  1. Department of Biomedical Engineering, Boston University, 44 Cummington Mall, Boston, MA, 02215, USA

    Lena Landaverde, Winnie Wong, Gabriela Hernandez, Andy Fan & Catherine Klapperich

Authors
  1. Lena Landaverde
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  2. Winnie Wong
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  3. Gabriela Hernandez
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  4. Andy Fan
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  5. Catherine Klapperich
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Corresponding author

Correspondence to Catherine Klapperich.

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Statement of ethical approval

All patient samples were acquired through the Biospecimen Archive Research Core (BARC) at Boston University Medical Center. These samples are collected from medical procedures performed at the Boston Medical Center (BMC). The use of the discarded human cervical swab samples was approved by the Boston University Institutional Review Board under protocol #3231.

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The authors declare that they have no conflicts of interest.

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Landaverde, L., Wong, W., Hernandez, G. et al. Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16. Anal Bioanal Chem 412, 6199–6209 (2020). https://doi.org/10.1007/s00216-020-02702-9

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  • DOI: https://doi.org/10.1007/s00216-020-02702-9

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