Detection of SARS-CoV-2 spike protein D614G mutation using µTGGE.

BACKGROUND: The accurate and expeditious detection of SARS-CoV-2 mutations is critical for monitoring viral evolution, assessing its impact on transmission, virulence, and vaccine efficacy, and formulating public health interventions. In this study, a detection system utilizing micro temperature gradient gel electrophoresis (µTGGE) was developed for the identification of the D614 and G614 variants of the SARS-CoV-2 spike protein. METHODS: The in vitro synthesized D614 and G614 gene fragments of the SARS-CoV-2 spike protein were amplified via polymerase chain reaction and subjected to µTGGE analysis. RESULTS: The migration patterns exhibited by the D614 and G614 variants on the polyacrylamide gel were distinctly dissimilar and readily discernible by µTGGE. In particular, the mid-melting pattern of D614 was shorter than that of G614. CONCLUSIONS: Our results demonstrate the capability of µTGGE for the rapid, precise, and cost-effective detection of SARS-CoV-2 spike protein D614 and G614 variants without the need for sequencing. Therefore, this approach holds considerable potential for use in point-of-care mutation assays for SARS-CoV-2 and other pathogens.

Biophysical Properties of the Fibril Structure of the Toxic Conformer of Amyloid-ß42: Characterization by Atomic Force Microscopy in Liquid and Molecular Docking.

Alzheimer's disease is associated with the aggregation of the misfolded neuronal peptide, amyloid-ß42 (Aß42). Evidence has suggested that several reasons are responsible for the toxicity caused by the aggregation of Aß42, including the conformational restriction of Aß42. In this study, one of the toxic conformers of Aß42, which contains a Glu-to-Pro substitution (E22P-Aß42), was explored using atomic force microscopy and molecular docking to study the aggregation dynamics. We proposed a systematic model of fibril formation to better understand the molecular basis of conformational transitions in the Aß42 species. Our results demonstrated the formation of amorphous aggregates in E22P-Aß42 that are stem-based, network-like structures, while the formation of mature fibrils occurred in the less toxic conformer of Aß42, E22-Aß42, that are sphere-like flexible structures. A comparison was made between the biophysical properties of E22P-Aß42 and E22-Aß42 that revealed that E22P-Aß42 had greater stiffness, dihedral angle, number of ß sheets involved, and elasticity, compared with E22-Aß42. These findings will have considerable implications toward our understanding of the structural basis of the toxicity caused by conformational diversity in Aß42 species.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses.

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.

DEP-On-Go for Simultaneous Sensing of Multiple Heavy Metals Pollutants in Environmental Samples.

We describe a simple and affordable "Disposable electrode printed (DEP)-On-Go" sensing platform for the rapid on-site monitoring of trace heavy metal pollutants in environmental samples for early warning by developing a mobile electrochemical device composed of palm-sized potentiostat and disposable unmodified screen-printed electrode chips. We present the analytical performance of our device for the sensitive detection of major heavy metal ions, namely, mercury, cadmium, lead, arsenic, zinc, and copper with detection limits of 1.5, 2.6, 4.0, 5.0, 14.4, and, 15.5 µg·L-1, respectively. Importantly, the utility of this device is extended to detect multiple heavy metals simultaneously with well-defined voltammograms and similar sensitivity. Finally, "DEP-On-Go" was successfully applied to detect heavy metals in real environmental samples from groundwater, tap water, house dust, soil, and industry-processed rice and noodle foods. We evaluated the efficiency of this system with a linear correlation through inductively coupled plasma mass spectrometry, and the results suggested that this system can be reliable for on-site screening purposes. On-field applications using real samples of groundwater for drinking in the northern parts of India support the easy-to-detect, low-cost (<1 USD), rapid (within 5 min), and reliable detection limit (ppb levels) performance of our device for the on-site detection and monitoring of multiple heavy metals in resource-limited settings.