Developing Paper Based Assay for Direct to LAMP Detection of Lyme Disease from Ticks

Saturday, October 26, 2024

12:50 PM - 12:55 PM EST

Location: Room 342

Presenting Author(s)

Kendal R. Phinney (she/her/hers)

Undergraduate Research Assitant
Erickson Lab
Endicott, New York, United States

Co-Author(s)

BF

Brooke Filanoski (she/her/hers)

Graduate Student
Cornell University
Ithaca, New York, United States

Primary Investigator(s)

DE

David Erickson

S.C. Thomas Sze Director & Sibley College Professor of Mechanical and Aerospace Engineering
Cornell University
Ithaca, New York, United States

Introduction: Loop-mediated isothermal amplification (LAMP) is a rapid and specific technique for nucleic acid amplification, and more feasible than polymerase chain reaction (PCR) in the point-of-care setting. LAMP can be conducted isothermally in less than an hour, requiring far less sophisticated lab equipment than PCR. This approach allows for quick disease quantification in limited-resource settings. However, persisting challenges such as transportation, storage, and pipetting of LAMP reagents requires -20 C storage and meticulous pipetting of reagents. We propose a paper-based assay with a pre-dried master mix for simple end-user operation. We chose the colorimetric metal ion indicator, hydroxynaphthol blue (HNB). HNB provides real-time and end-point LAMP results due to autofluorescence and color change from purple to blue as a result of free magnesium ion change throughout the LAMP reaction. Through testing a combination of reagent ratios and paper types, we are creating a visually indicative test that will aid in the detection of Lyme disease directly from tick samples. This system will allow for ticks to be collected from humans, animals, or natural areas and be flagged for potential health exposure to the bacteria Borrelia. We are optimizing our procedure so that simple in-field detection is affordable and accessible. These outcomes exhibit our current results with paper-based results closely following.

Materials and

Methods: The LAMP master mix includes MgSO4, isothermal buffer, dNTPs, primers, hydroxynaphthol blue (HNB), trehalose, and Bst polymerase. Here, we outline varying reagent combinations to dry together before adding additional reagents (if any) for final amplification. The combinations were dried, rehydrated, and heated. For drying, the tubes were placed in an oven at 37 C for 48 hours. Dried reagents were reconstituted with DNase/ RNase free water and any LAMP reagents that were not dried were added. We used published SARS-CoV-2 primers and positive control for preliminary results since we previously reliably used them in-lab; non-template control DNA was used for the negative control. The LAMP reaction was isothermally heated at 65 C for 50 minutes. We measured real-time fluorescence and observed end-point colorimetric results to determine whether the reagent combinations could be translated to paper.

To test paper membranes, we used a standard, sterilized hole puncher to hole punch papers including Whatman grade cellulose, nitrocellulose, and glass fiber membranes. These membranes were added to a 96 well plate and reagents were added in varying combinations and ratios to produce similar colors to the liquid reagents in the PCR tubes. This color was tuned so that slight changes in magnesium that occur during LAMP reactions would change the paper membrane from purple to blue in the presence of viral amplification. From here, we intend to test our PCR tube results on the paper membranes in the near future.

Results, Conclusions, and Discussions: Through real-time and end-point results, we were able to produce dried LAMP master mix results that compare to fresh master mix. These results include discernible differences between positive and negative amplification using the HNB indicator, which can be seen in Figure 1. The presence of target DNA results in an HNB shift from purple (starting color) to sky blue (positive reaction color). This color change also results in an auto-fluorescence change, ideally measured at 540 excitation and 610 emission wavelengths, but can be captured in the blue and red channels as shown in Figure 2. The threshold cycle (Ct) time was extracted using our lab’s in-house algorithm, and graphed alongside reagent combinations in Figure 3. This data tentatively outlines what can be translated to paper membranes. Preliminary results, pictured in Figure 5, show the purple to blue color change of the paper membranes. These LAMP dots will enable easy detection of Borrelia that causes Lyme disease.

Overall, these results highlight that certain reagents can be combined and dried ahead of time to simplify steps and technology required of the end user. Even though the N gene from SARS-CoV2 was used in this instance, we believe there is promise for our future direction of adapting this assay to the glpQ gene of the Borrelia species as a positive control to detect relapsing fever Borrelia. We are currently developing a positive control and primer specific assay to detect and diagnose tick-borne relapsing fever in the North Eastern United States. Looking at Figure 5, DNA will be extracted from a tick when removed from an individual and added to a cellulose paper membrane, which contains the dried LAMP master mix. These membranes will be incubated and analyzed with the naked eye or a plate reader. This new technology for diagnosing samples could be used in an array of point-of-care applications going forward.

Acknowledgements (Optional):