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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster F5 - One-Pot Ligation Loop-Mediated Isothermal Amplification (LAMP) assay to rapidly and sensitively detect miRNA biomarkers in solution

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Alie Bogusch

Undergraduate Researcher
University of Maryland, College Park
West Chester, Pennsylvania, United States

Primary Investigator(s)

IW

Ian M. White, PhD (he/him/his)

Professor
University of Maryland
College Park, Maryland, United States

Co-Author(s)

EB

Evan Benke

Graduate Student
University of Maryland, United States

Introduction: Rapid diagnostics are needed to detect and monitor life-threatening diseases, such as cancer and auto-immune disorders. MicroRNA (miRNA) biomarkers are elevated in the bloodstream in the case of such diseases [1]. miRNAs are traditionally detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR), but this method approach requires additional steps because the miRNA is too short for standard PCR. Ligation Loop-Mediated Isothermal Amplification (Ligation LAMP) is an alternative to RT-qPCR that can detect small amounts of miRNA with fewer assay steps. However, ligation LAMP is a two step process that requires access to a lab and technical skills like micropipetting [2]. Here, we explore the use of Thermally Responsive Alkane Partitions (TRAPS) to minimize the need for hands-on precise assay steps in a single tube ligation LAMP reaction [3]. TRAPs are alkane layers that serve as solid partitions for reagents at ambient temperature and that breach when warmed above the melt temperature, mixing the initially partitioned reagents (thus eliminating manual pipetting steps). The use of TRAPS simplifies the ligation LAMP workflow, making it more point-of-care friendly. This study focuses on the detection of miRNA-155, a cancer biomarker. We demonstrate comparable assay speed and sensitivity between the 2-pot and TRAP-enabled 1-pot versions of ligation LAMP, showing that assay performance is not sacrificed with the single-step approach. The 1-pot Ligation LAMP assay may enable the development of future point-of-care diagnostic devices that can allow patients to be rapidly and accurately diagnosed on site, without the need to send samples to a lab.

Materials and

Methods: In a 2-pot ligation LAMP system the 2 steps proceed separately. First ligation occurs, where two DNA hairpin primers specifically hybridize with the miRNA target, splinting the two hairpins together. SplintR ligase then permanently joins the two hairpins together, forming the double hairpin DNA strand. In the second step, LAMP reagents are added to the completed ligation reaction and the double hairpin is amplified with LAMP primers and BST 2.0 warmstart polymerase (New England Biolabs).
The One-Pot Ligation LAMP assay is assembled in two liquid layers, with a hexacosane TRAP (Tm=56°C) separating the two steps in a single PCR tube (Figure 1A). When the reaction is fully assembled in the tube, it can proceed semi-automatically. First, the reaction is incubated at 40°C to allow ligation of the hairpin structure, then the temperature is raised to 63°C, melting the hexacosane TRAP, mixing the reagents, and allowing LAMP to proceed. The TRAP geometry needs to cover the entire bottom liquid layer and touch the edges of the tube. Its hydrophobic nature and low density as a liquid ensures that it does not mix with the reaction once melted, instead floating to the top of the tube. The reaction progress was measured using an intercalating dye and real time fluorescence measurements in a thermocycler. The assay was run with various concentrations of miRNA-155 and then compared to the same reaction and concentrations from a 2 pot system.

Results, Conclusions, and Discussions: The 1-pot assay was found to have comparable speed and sensitivity to the 2 -ot system, without the need of an intermediate pipetting step, as shown in Figure 1-B. Real time fluorescent LAMP was used to test its sensitivity to different concentrations of miRNA-155. Increasing fluorescence indicates amplification of miRNA-155. The concentration of miRNA was detectable from 10 nM to as low as 10 fM. In the 2-pot system, amplification of the highest concentration (100 nM) starts amplifying consistently by about 25 minutes and the NTC does not start before 50 minutes (Figure 1C). The 1 pot system is slightly slower, with the 100 nM concentration beginning amplification at 35 minutes and the NTC starting at 80 minutes. However, the 1 pot system shows separation between each of the concentrations and the NTC consistently amplified after the targets, with a detection limit of about 1 fM, showing how it is sensitive to the miRNA-155 target. It also shows good reproducibility, which indicates consistent melting of the TRAP and thus a consistent release and mixing of the reagents.

Conclusion:
The promising results of the assay indicate that ligation LAMP can be performed in a single tube without the need for intermediate pipetting steps with comparable speed and sensitivity to the same reaction in a 2-pot system. We expect this to lead to point-of-care implementations of ligation LAMP for the detection of miRNA, which can ultimately allow quicker diagnoses and better outcomes.

Acknowledgements (Optional):

References:
[1] J. Wang, J. Chen, and S. Sen, “MicroRNA as Biomarkers and Diagnostics,” Journal Cellular Physiology, vol. 231, no. 1, pp. 25–30, Jan. 2016.
[2] W. Du, M. Lv, J. Li, R. Yu, and J. Jiang, “A ligation-based loop-mediated isothermal amplification (ligation-LAMP) strategy for highly selective microRNA detection,” Chem. Commun., vol. 52, no. 86, pp. 12721–12724, 2016.
[3] D. J. Boegner, M. L. Everitt, and I. M. White, “Thermally Responsive Alkane Partitions for Assay Automation,” ACS Appl. Mater. Interfaces, vol. 14, no. 7, pp. 8865–8875, Feb. 2022.