Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples

Abstract

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader’s analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2 750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.

Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid

“…Rapid molecular biosensing is an emerging application area for synthetic biology, particularly for the on-demand detection of viruses 1-5 microbes 6,7 , metals 8,9 , and pollutants 10,11 in biological or environmental samples. Traditional methods for detecting these analytes of interest require collection of large-volume samples on-site and transportation to centralized laboratories equipped with sophisticated analytical equipment.…”

Fully Dried Two-Dimensional Paper Network for Enzymatically Enhanced Detection of Nucleic Acid Amplicons

“…A previous study showed that the visible threshold for an untrained user without instruction was quantified at 0.02 grayscale intensity when normalized to the background using a custom MATLAB program. 21,22 Based on this, we used the 0.02 grayscale intensity as the visible threshold for a positive result in our experiments.…”

“…Test and control line intensities were analyzed with a custom MATLAB script. 21,22 The visible threshold (the visual cutoff for interpreting a test line as positive) of the background normalized grayscale intensity was set to 0.02 for all signal analyses according to Phillips et al 22 For LOD tests, dilutions of 100 μM probe 3 solution in PBST were used. Tests were run on assembled 2DPNs stored between 1 and 7 days.…”

“…28 If we assume a rectangular crosssection of nitrocellulose and that particles are diluted in water, then P c = γ(2 cos (θ)/h + 2 cos (θ)/w), where every surface i is nitrocellulose and θ is the advancing contact angle of pure water on nitrocellulose. Given that our nitrocellulose membrane, FF80HP (GE Healthcare), has pore size comparable to HF135 (Millipore), 21 which can pass particles of up to 0.5 μm, 29 we can assume an approximate pore size of 0.5 μm. For water on 0.45 μm pore size nitrocellulose, the advancing contact angle is measured to be approximately 40°.…”

Microfluidic Rapid and Autonomous Analytical Device (microRAAD) to Detect HIV from Whole Blood Samples

“…One such isothermal amplification method, loop-mediated isothermal amplification (LAMP), provides specific and efficient amplification of target nucleic acids by targeting 8 unique sequences (Nagamine et al, 2002). Operable at a single temperature (most efficiently between 65 and 72 °C) (NEB, n.d.; Rolando et al, 2019), LAMP robustly amplifies even in the presence of complex sample matrices, further reducing sample processing and instrumentation requirements (Clayton et al, 2019;Mori and Notomi, 2009;Phillips et al, 2018). To expedite sample preparation steps, such as reverse transcription (RT) of HIV RNA targets, several groups have demonstrated that RT can be performed using the same assay conditions as LAMP (Curtis et al, 2012;Damhorst et al, 2015;Odari et al, 2015;Rudolph et al, 2015).…”