148. Single-amplicon, Multiplex Real-time RT-PCR with Tiled Probes to Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

Background Detection and surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of great public health importance. Broadly accessible and inexpensive assays are needed to enhance variant surveillance and detection globally. We developed and validated a single-reaction multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations associated with variants of concern (VOC). Methods A single primer pair was designed to amplify a 348 bp region of spike. Probes were initially designed with locked nucleic acids (LNAs) to increase probe melting temperature, shorten probe length, and specifically detect 417K, E484K, and N501Y (Figure). The assay was optimized and evaluated using characterized variant sample pools. Clinical evaluation was performed on a convenience set of residual nasopharyngeal swabs, and variant calls were confirmed by SARS-CoV-2 genomic sequencing in a subset of samples. Following the initial evaluation, unmodified probes (without LNAs) were designed to detect L452R, L452Q, and E484Q. Figure. Spike SNP distinguishes mutations occurring in different lineages (A-C). Representative results of variant detection a single Spike SNP run are shown for mutations in the codons for 4177K (A) and mutations that encode 484K (B) and 501Y (C). Curves show dilutions of the following variants: blue, BEI 52286 (wild type); pink B.1.1.7; purple, B1.525; and green, P.1. Variant pools were used for B.1.17, B.1.525, and P.1 strains. Curves are displayed for a given dilution in each channel and result interpretation is shown (D). Results The lower limit of 95% detection was 2.46 to 2.48 log10 GE/mL for the three targets (~1-2 GE/reaction). Among 253 nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%), including all samples with Ct values < 30 (220/220) for the N2 target and 18/33 samples with N2 Ct values ≥ 30. Results were confirmed by SARS-CoV-2 genomic sequencing in 50/50 samples (100%). Subsequent addition of the 452R probe did not affect performance for the original targets, and probes for 452Q and 484Q performed similarly to LNA-modified probes. Conclusion The Spike SNP assay provides fast, inexpensive and sensitive detection of specific mutations associated with SARS-CoV-2 VOCs, and the assay can be quickly modified to detect new mutations in the receptor binding domain. Similar analytical performance of LNA-modified and unmodified probes presents options for future assay customization that balance the shorter probe length (LNAs) and increased accessibility (unmodified). The Spike SNP assay, if implemented across laboratories offering SARS-CoV-2 testing, could greatly increase capacity for variant detection and surveillance globally. Disclosures Colleen S. Kraft, MD, MSc, Rebiotix (Individual(s) Involved: Self): Advisor or Review Panel member

Modelling the Spectral Shape of Continuous-Wave Lidar Measurements in a Turbulent Wind Tunnel

This paper describes the development of a model for the turbulence spectrum measured by a short-range, continuous-wave lidar. The lidar performance was assessed by measurements conducted with two WindScanners in an open jet wind tunnel equipped with an active grid, for a range of different turbulent wind conditions. A one-dimensional hot wire anemometer was used as a reference for characterising the lidar turbulence measurement. In addition to addressing the statistics, the correlation between the time series and the mean error on the wind speed, the lidar measurement turbulence spectrum is compared with a theoretical spectrum using Taylor's frozen turbulence hypothesis. A theoretical model for the probe length averaging effect is applied in the frequency domain, using a Lorentzian filter in combination with generated white noise, and evaluated by qualitatively matching the lidar measurement spectrum. High goodness of fit coefficients and low mean absolute errors between hot wire and WindScanner were observed for the measured time series. The correlation showed an inverse relationship with the prevalent turbulence intensity in the flow for cases with a comparable power spectrum shape. Larger flow structures can be captured more accurately by the lidar, whereas small-scale turbulent flow structures are partly filtered out as a result of the lidars' probe volume averaging. It is demonstrated that an accurate way to define the frequency at which the lidar power spectrum starts to deviate from the hot wire reference spectrum is the point at which the coherence drops below 0.5. This coherence-based cut-off frequency increases linearly with the mean wind speed and is generally an order of magnitude lower than the probe length cut-off frequency, estimated according to a simple model based on Taylor's frozen turbulence hypothesis. A convincing match between the modelled and the actual WindScanner power spectrum was found for various different cases, which confirmed that the deviation of the lidar measurement power spectrum in the higher frequency range can be analytically explained and modelled as a combination of a Lorentzian probe length averaging effect and white noise in the lidar measurement.

Spectral correction of turbulent energy damping on wind lidar measurements due to spatial averaging

Continuous advancements in pulsed wind lidar technology have enabled compelling wind turbulence measurements within the atmospheric boundary layer with probe lengths shorter than 20 m and sampling frequency on the order of 10 Hz. However, estimates of the radial velocity from the back-scattered lidar signal are inevitably affected by an averaging process within each probe volume, generally modeled as a convolution between the true velocity projected along the lidar line-of-sight and an unknown weighting function representing the energy distribution of the laser pulse along the probe length. As a result, the spectral energy of the turbulent velocity fluctuations is damped within the inertial subrange, thus not allowing one to take advantage of the achieved spatio-temporal resolution of the lidar technology. We propose to correct the turbulent energy damping on the lidar measurements by reversing the effect of a low-pass filter, which can be estimated directly from the power spectral density of the along-beam velocity component. Lidar data acquired from three different field campaigns are analyzed to describe the proposed technique, investigate the variability of the filter parameters and, for one dataset, assess the corrected velocity variance against sonic anemometer data. It is found that the order of the low-pass filter used for modeling the energy damping on the lidar velocity measurements has negligible effects on the correction of the second-order statistics of the wind velocity. In contrast, the cutoff wavenumber plays a significant role in spectral correction encompassing the smoothing effects connected with the lidar probe length. Furthermore, the variability of the spatial averaging on wind lidar measurements is investigated for different wind speed, turbulence intensity, and sampling height. The results confirm that the effects of spatial averaging are enhanced with decreasing wind speed, smaller integral length scale and, thus, for smaller sampling height. The method proposed for the correction of the second-order turbulent statistics of wind-velocity lidar data is a compelling alternative to existing methods because it does not require any input related to the technical specifications of the used lidar system, such as the energy distribution over the laser pulse and lidar probe length. On the other hand, the proposed method assumes that surface-layer similarity holds.

Quantifying the binding between proteins and open chromatin-like DNA sequences with gold nanorods

We developed a gold nanorod-based colorimetric assay for the binding of transcription factors to DNA in long open chromatin-like structures. After determining of the binding affinity and stoichiometry, we explored the effect of the probe length on the assay performance.

Characterizing the non-crosslinked aggregation of DNA-modified gold nanoparticles: effects of DNA length and terminal base pair

We investigated the effects of particle size, temperature, electrolyte concentration, and probe length on the non-crosslinked aggregation of DNA-modified GNPs.