A smartphone-based platform for ratiometric visualization of SARS-CoV-2 via an oligonucleotide probe.

COVID-19 necessitates the development of reliable and convenient diagnostic tools. In this work, a facile 3D-printed smartphone platform was constructed that achieved reliable visual detection of SARS-CoV-2 by eliminating the effect of ambient light and fixing the camera position relative to the sample. The oligonucleotide probe is modified with orange-red-emitting TAMRA working as an internal standard and green-emitting FAM serving as a sensitive sensing agent. Under 365-nm UV excitation, the emission wavelengths of TAMRA and FAM are 580 nm and 518 nm, respectively. When the probes interact with the targets, the green fluorescence gradually restores while the orange-red fluorescence remains stable. Thus, a striking color transition from orange-red to green could be observed by the naked eye. The detection limit of SARS-CoV-2 nucleic acid is 0.23 nM, and the entire process of color change could be completed in 25 min. Furthermore, the RGB value analysis of the sample solution was conducted using a smartphone for reliable and reproducible discrimination of SARS-CoV-2. The proposed smartphone platform might establish a general method for visual detection of SARS-CoV-2 nucleic acid as well as other virus-related diseases.

A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview.

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.

BACKGROUND: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. METHODS: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. RESULTS: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction. CONCLUSIONS: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.

Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry.

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection.