A Simple, Pipette-free, and Power-free Device for Virus Nucleic Acid Detection

A portable, inexpensive, and easy-to-manufacture microfluidic device is developed for the detection of SARS-CoV-2 dsDNA fragments. In this device, four reaction chambers separated by carbon fiber rods are pre-loaded with isothermal amplification and CRISPR-Cas12a reagents. The reaction is carried out by simply pulling the rods, without the need for manual pipetting. To facilitate power-free pathogen detection, the entire detection is designed to be heated with a disposable hand warmer. After the CRISPR reaction, the fluorescence signal generated by positive samples is identified by naked eye, using an inexpensive flashlight. This simple and sensitive device will serve as a new model for the next-generation viral diagnostics in either hospital or resource-limited settings. © 2023 SPIE.

Asthenopia prevalence and vision impairment severity among students attending online classes in low-income areas of western China during the COVID-19 pandemic.

INTRODUCTION: This study explored the impact of online learning during the coronavirus disease 2019 (COVID-19) pandemic on asthenopia and vision impairment in students, with the aim of establishing a theoretical basis for preventive approaches to vision health. METHODS: This balanced panel study enrolled students from western rural China. Participant information was collected before and during the COVID-19 pandemic via questionnaires administered at local vision care centres, along with clinical assessments of visual acuity. Paired t tests and fixed-effects models were used to analyse pandemic-related differences in visual status. RESULTS: In total, 128 students were included (mean age before pandemic, 11.82 ± 1.46 years). The mean total screen time was 3.22 ± 2.90 hours per day during the pandemic, whereas it was 1.97 ± 1.90 hours per day in the pre-pandemic period (P<0.001). Asthenopia prevalence was 55% (71/128) during the pandemic, and the mean visual acuity was 0.81 ± 0.30 logarithm of the minimum angle of resolution; these findings indicated increasing vision impairment, compared with the pre-pandemic period (both P<0.001). Notably, asthenopia prevalence increased by two- to three-fold, compared with the pre-pandemic period. An increase in screen time while learning was associated with an increase in asthenopia prevalence (P=0.034). CONCLUSION: During the COVID-19 pandemic, students spent more time on online classes, leading to worse visual acuity and vision health. Students in this study reported a significant increase in screen time, which was associated with increasing asthenopia prevalence and worse vision impairment. Further research is needed regarding the link between online classes and vision problems.

Comprehensive, longitudinal wastewater surveillance for SARS-CoV-2 across a university campus, demonstrates low levels of SARS-CoV-2 activity relative to the surrounding community

Background. Universities are interactive communities where frequent contacts between individuals occur, increasing the risk of outbreaks of COVID-19. We embarked upon a real-time wastewater (WW) monitoring program across the University of Calgary (UofC) campus measuring WW SARS-CoV-2 burden relative to levels of disease in the broader surrounding community. Figure 1 The colour scheme shows 6 sewer sub-catchments at the University of Calgary. Auto samplers were deployed at 4 sampling nodes within sub-catchments CR and YA (both residence halls), and UCE and UCS (catchments that include several campus buildings). Figure 2 Log10-transformed abundance (i.e., copies per mL) of nucleocapsid gene (i.e., N1) for SARS-CoV-2 for each sampling location during October 2021 - April 2022. Locations denoted by the same letters (A, B, or C) show no statistical difference (p > 0.05) according to the Wilcoxon rank-sum test. The WWTP sample corresponds to a catchment area covering most of Calgary including the university campus, for which sampling locations CR, UCE, UCS, and UCW are defined in Fig. 1. Methods. From October 2021 - April 2022, WW was collected thrice weekly across UofC campus through 4 individual sewer sampling nodes (Fig. 1) using autosamplers (C.E.C. Analytics, CA). Results from these 4 nodes were compared with community monitoring at Calgary's largest WW treatment plant (WWTP), which received WW from surrounding neighborhoods, and also from UofC. Nucleic acid was extracted from WW for RTqPCR quantification of the N1 nucleocapside gene from SARS-CoV-2 genomic RNA. Qualitative (positive samples defined if cycle threshold < 40) and quantitative statistical analyses were performed using R. Results. Levels of SARS-CoV-2 in WW were significantly lower at all campus monitoring sites relative to the WWTP (Wilcoxon rank-sum test p < 0.05;Fig. 2). The proportion of WW samples that were positive for SARS-CoV-2 was significantly higher for WWTP than at least two campus locations (p < 0.05 for Crowsnest Hall and UCE - University way and campus drive) according to Fischer's exact 2-sided test. The proportion of WW samples with positive WW signals were still higher for WWTP than the other two locations, but statistically not significant (p = 0.216). Among campus locations, the buildings in UCE catchment showed much lower N1 signals than other catchments, likely owing to buildings in this catchment primarily being administration and classroom environments, with lower human-to-human contact and less defecation compared to the other 3 catchments, which include residence hall, a dining area, and/or laboratory spaces. Conclusion. Our results show that SARS-CoV-2 RNA shedding in WW at the U of C is significantly lower than the city-wide signal associated with surrounding neighborhoods. Furthermore, we demonstrate that WW testing at well-defined nodes is a sampling strategy for potentially locating specific places where high transmission of infectious disease occurs.

Comparative performance of RTqPCR vs RTddPCR for the detection of SARS-CoV-2 in wastewater (WW) collected from a range of sites and scales across the sewer network of Calgary, Alberta

Background. We sought to compareWWSARS-CoV-2 RNA detection across a range of sites and scales using RTqPCR and RTddPCR. Figure. Methods. Composite-24hWW was collected from aWWtreatment plant (WTP;n=18), a neighborhood (Nb1;n=12) and three hospitals;H-1, H-2, and H-3 (3-sites;A-C)(n=84). RNA was extracted using the 4S-silica column method. RTqPCR (QuantStudio5, Thermo Fisher) and RTddPCR (C1000 Thermal Cycler and QX200 Droplet Reader, BioRad) quantified SARS-CoV-2 RNA nucleocapsid (N2, US CDC) and envelope (E Sarbeco, Corman et al 2020) in triplicate. Fisher's exact test was used to compare assay sensitivity. Correlations between modalities and RNA - clinically-confirmed COVID-19 cases (defined by postal code of primary residence using 5-day rolling average) was assessed using Persons correlation. Results. 114 samples were tested (02/23/2021-04/22/2021). SARS-CoV-2-N2 was identified in 90/114 (79%) by RTqPCR and 89/114 (78%) by ddPCR (p=1). SARS-CoV-2 E was found in 72/114 (63%) by RTqPCR and 90/114 (79%) by ddPCR, p=0.01. Correlations between modalities were strongest for N2 relative to E across all sites (see Table). N2 correlated with clinically diagnosed cases for both modalities greater at the level of the WTP (RTqPCR;r=0.8972, p< 0.0001and ddPCR;0.933, p< 0.0001) relative to neighborhood (RTqPCR;r=0.6, p=0.04 and ddPCR;0.60, p=0.04). E correlated to a lesser degree with cases at WTP (RTqPCR;r=0.65, p=0.0035 and ddPCR;0.88, p=< 0.001) and neighborhoods (RTqPCR;r=0.40, p=0.20 and ddPCR;r=0.43, p=0.16). Conclusion. SARS-CoV-2 detection of N2 was similar between RTqPCR and RTddPCR across a range of sites and scales in the sewershed, and this correlated best with clinical cases whereas E detection was superior with ddPCR.

Surveillance of SARS-CoV-2 variants of concern (VOC) in hospital wastewater (WW) and its correlation with hospitalized cases of COVID-19 and the occurrence of outbreaks

Background. WW surveillance enables real time monitoring of SARS-CoV-2 burden in defined sewer catchment areas. Here, we assessed the occurrence of total, Delta and Omicron SARS-CoV-2 RNA in sewage from three tertiary-care hospitals in Calgary, Canada. Methods. Nucleic acid was extracted from hospital (H) WW using the 4S-silica column method. H-1 and H-2 were assessed via a single autosampler whereas H-3 required three separate monitoring devices (a-c). SARS-CoV-2 RNA was quantified using two RT-qPCR approaches targeting the nucleocapsid gene;N1 and N200 assays, and the R203K/G204R and R203M mutations. Assays were positive if Cq< 40. Cross-correlation function analyses (CCF) was performed to determine the timelagged relationships betweenWWsignal and clinical cases. SARS-CoV-2 RNA abundance was compared to total hospitalized cases, nosocomial-acquired cases, and outbreaks. Statistical analyses were conducted using R. Results. Ninety-six percent (188/196) of WW samples collected between Aug/ 21-Jan/22 were positive for SARS-CoV-2. Omicron rapidly supplanted Delta by mid-December and this correlated with lack of Delta-associated H-transmissions during a period of frequent outbreaks. The CCF analysis showed a positive autocorrelation between the RNA concentration and total cases, where the most dominant cross correlations occurred between -3 and 0 lags (weeks) (Cross-correlation values: 0.75, 0.579, 0.608, 0.528 and 0.746 for H-1, H-2, H-3a, H-3b and H-3c;respectively). VOC-specific assessments showed this positive association only to hold true for Omicron across all hospitals (cross-correlation occurred at lags -2 and 0, CFF value range between 0.648 -0.984). We observed a significant difference in median copies/ ml SARS-CoV-2 N-1 between outbreak-free periods vs outbreaks for H-1 (46 [IQR: 11-150] vs 742 [IQR: 162-1176], P< 0.0001), H-2 (24 [IQR: 6-167] vs 214 [IQR: 57-560], P=0.009) and H-3c (2.32 [IQR: 0-19] vs 129 [IQR: 14-274], P=0.001). Conclusion. WWsurveillance is a powerful tool for early detection andmonitoring of circulating SARS-CoV-2VOCs.Total SARS-CoV-2 andVOC-specificWWsignal correlated with hospitalized prevalent cases of COVID-19 and outbreak occurrence.

Theoretical Study on Raman Characteristic Peaks of Coronavirus Spike Protein Based on Deep Learning

COVID-19, which has lasted for a year, has caused great damage to the global economy. In order to control COVID-19 effectively, rapid detection of COVID-19 (SARS-CoV-2) is an urgent problem. Spike protein is the detection point of Raman spectroscopy to detect SARS-CoV-2. The construction of spike protein Raman characteristic peaks plays an important role in the rapid detection of SARS-CoV-2 using Raman technology. In this paper, we used Deep Neural Networks to construct the amide I and III characteristic peak model of spike proteins based on simplified exciton model, and combined with the experimental structures of seven coronaviruses (HCoV-229E, HCoV-HKUl, HCoV-NL63, HCoV-OC43, MERS-CoV, SARS-CoV, SARS-CoV-2) spike proteins, analyzed the differences of amide I and III characteristic peaks of seven coronaviruses. The results showed that seven coronaviruses could be divided into four groups according to the amide I and III characteristic peaks of spike proteins: SARS-CoV-2, SARS-CoV, MERS-CoV form a group;HCoV-HKUl, HCoV-NL63 form a group;HCoV-229E and HCoV-OC43 form a group independently. The frequency of amide I and III in the same group is relatively close,and it is difficult to distinguish spike proteins by the frequency of amide I and III ;the characteristic peaks of amide I and III in different groups are quite different, and spike proteins can be distinguished by Raman spectroscopy. The results provide a theoretical basis for the development of Raman spectroscopy for rapid detection of SARS-CoV-2. © 2022 Science Press. All rights reserved.

Recent advances in nucleic acid analysis and detection with microfluidic and nanofluidics

Microfluidic- and nanofluidics-based nucleic acid sensing and analysis have become of interest to the public, especially during the current COVID pandemic. In this chapter, we provide a comprehensive review of recent research dedicated to the advances of nucleic acid analysis and detection including various polymerase chain reaction platforms, isothermal target amplification methods, and emerging amplification-free methods, such as optofluidics sensing, electrochemical sensing, thermal sensing, and advanced microscopy for label-free DNA/RNA analysis. The future advancement and prospects of nucleic acid analysis are also discussed. © 2022 Elsevier B.V. All rights reserved.

Surface-enhanced Raman effect of new coronavirus S protein in gold nanoparticles

Surface-enhanced Raman spectroscopy (SERS) technology has been widely used in viral molecular detection due to its high sensitivity, simple operation and rapid detection. The research of virus detection by Raman technology at home and abroad mainly focuses on the detection of the SERS spectrum of viral nucleic acids and various bases that make up the nucleic acids, and detection of viral proteins is rare. In this paper, the S protein of the new coronavirus (SARS-CoV-2) is used as the detection object, and with the label-free SERS detection method, the ordinary Raman spectra of solid and saturated liquid S protein of the SARS-CoV-2 and the SERS spectra of the low-concentration S protein of SARS-CoV-2 on the substrate of gold nanoparticles with a size of 40 nm are compared. The results show that it is completely feasible to use SERS technology to detect the S protein of SARS-CoV-2 on the substrate of 40 nm gold nanoparticles. The carboxyl groups in the S protein molecule of SARS-CoV-2 and gold nanoparticles are molecularly enhanced, and the amino groups and gold nanoparticles are electromagnetically enhanced, so that the Raman effect of the S protein of the SARS-CoV-2 is enhanced and the peak position is moved to a certain extent. The experiments obtained relatively good SERS spectra of the low-concentration S protein of SARS-CoV-2, which provides a method for the establishment of a sensitive, specific and rapid detection technology for the S protein of the SARS-CoV-2. © 2021, Editorial Office of High Power Laser and Particle Beams. All right reserved.

Viral nucleic acid detection with crispr-cas12a using high contrast cleavage detection on micro-ring resonator biosensors

Micro-ring resonators have emerged as a powerful platform for analyzing and detecting biomolecules at low concentrations. Here we demonstrate a high contrast cleavage detection (HCCD) assay on a micro-ring resonator to sense the cleavage of DNA reporters linked to high-contrast nanoparticles (NPs), leading to dramatic optical signal amplification. The HCCD mechanism is coupled with a CRISPR-Cas12a assay for rapid and sensitive on-chip nucleic acid detection. Leveraging high-contrast gold nanoparticle (AuNP) reporters, an -8 nm resonance shift is observed by using a 1 nM of complementary DNA (cDNA) target, matching part of the SARS-CoV-2 sequence. In addition, we show that a micro-ring resonator can not only record the entire surface functionalization process, as has been show previously, but also monitor CRISPR reactions in-situ. This work is the first step toward novel nucleic acid amplification-free detection via a combination of integrated photonics and CRISPR-Cas collateral cleavage assays. © 2021 SPIE. All rights reserved.