RNA purification-free detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Background Current diagnosis of SARS-CoV-2 infection relies on RNA purification prior to amplification. Typical extraction methods limit the processing speed and turnaround time for SARS-CoV-2 diagnostic testing. Methods Here, we applied reverse transcription loop-mediated isothermal amplification directly onto human clinical swabs samples to amplify the RNA from SARS-CoV-2 swab samples after processing with chelating resin. Results By testing our method on 64 samples, we managed to develop an RT-LAMP assay with 95.9% sensitivity (95% CI 86 to 99.5%) and 100% specificity (95% CI 78.2–100%). Conclusion The entire process including sample processing can be completed in approximately 50 min. This method has promising potential to be applied as a fast, simple and inexpensive diagnostic tool for the detection of SARS-CoV-2.

Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.

Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Background Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. Methods In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. Results Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9–96.1%) and 67.4% sensitive (95% CI: 51.5–80.9%) for E gene and RdRp gene, respectively. Conclusion Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

Clinical performance and potential of a SARS-CoV-2 detection kit without RNA purification steps

Objectives Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading globally. Early diagnosis plays an essential role in controlling the infection. Therefore, early and accurate SARS-CoV-2 detection assays along with easy operation are required. The aim of this study was to compare the clinical performance of the Ampdirect™ 2019-nCoV Detection Kit (SHIMADZU assay), which does not require RNA purification steps, with that of the preexisting SARS-CoV-2 detection assays, which use a purified RNA template. Methods A total of 71 samples (65 nasopharyngeal specimens and 6 sputum specimens) were collected from 32 individuals, including patients infected with SARS-CoV-2 and those with suspected infection. The sensitivity and kappa (κ) coefficient were assessed between the SARS-CoV-2 detection assays using the reference standard, which was defined as a true positive result by any one of the four SARS-CoV-2 detection assays. Results The overall sensitivity and κ coefficient of the SHIMADZU assay were 86.0% (95% confidence interval [CI]: 77.9–94.2) and 0.83 (95% CI: 0.69–0.96), respectively. In particular, among the 18 samples collected within 10 days from symptom onset, the sensitivity and κ coefficient of the SHIMADZU assay were 100% and 1.0, respectively. Conclusions Although a relatively small number of samples was evaluated, the SHIMADZU assay showed good analytical performance and as such would be highly useful for the detection of SARS-CoV-2. The test can be performed easily and quickly and has the potential for future applications in situations where a highly sensitive diagnosis is required.

A simple method for in situ, multiplexed measurement of RNA degradation by flow cytometry

RNA degradation plays a major role in cellular function, but current methods for measuring RNA degradation require RNA purification or are low throughput. Here we show how a flow-FISH assay can be used for high-throughput, in situ measurement of RNA degradation without RNA purification. We demonstrate how this approach can be used to simultaneously measure RNA degradation rates of different RNA sequences in a single assay and explore how the assay can be used to examine the effect of cellular context on RNA degradation rates. This assay will be generally useful to quantitatively measure how natural and engineered biological function depends on RNA half-life.

The Novel Non-coding Transcriptional Regulator Gm18840 Drives Cardiomyocyte Apoptosis in Myocardial Infarction Post Ischemia/Reperfusion

BackgroundIschemia/reperfusion-mediated myocardial infarction (MIRI) is a major pathological factor implicated in the progression of ischemic heart disease, but the key factors dysregulated during MIRI have not been fully elucidated, especially those essential non-coding factors required for cardiovascular development.MethodsA murine MIRI model and RNA sequencing (RNA-seq) were used to identify key lncRNAs after myocardial infarction. qRT-PCR was used to validate expression in cardiac muscle tissues and myocardial cells. The role of Gm18840 in HL-1 cell growth was determined by flow cytometry experiments in vitro. Full-length Gm18840 was identified by using a rapid amplification of cDNA ends (RACE) assay. The subcellular distribution of Gm18840 was examined by nuclear/cytoplasmic RNA fractionation and qRT-PCR. RNA pulldown and RNA immunoprecipitation (RIP)-qPCR assays were performed to identify Gm18840-interacting proteins. Chromatin isolation by RNA purification (ChIRP)-seq (chromatin isolation by RNA purification) was used to identify the genome-wide binding of Gm18840 to chromatin. The regulatory activity of Gm18840 in transcriptional regulation was examined by a luciferase reporter assay and qRT-PCR.ResultsGm18840 was upregulated after myocardial infarction in both in vivo and in vitro MIRI models. Gm18840 was 1,471 nt in length and localized in both the cytoplasm and the nucleus of HL-1 cells. Functional studies showed that the knockdown of Gm18840 promoted the apoptosis of HL-1 cells. Gm18840 directly interacts with histones, including H2B, highlighting a potential function in transcriptional regulation. Further ChIRP-seq and RNA-seq analyses showed that Gm18840 is directly bound to the cis-regulatory regions of genes involved in developmental processes, such as Junb, Rras2, and Bcl3.ConclusionGm18840, a novel transcriptional regulator, promoted the apoptosis of myocardial cells via direct transcriptional regulation of essential genes and might serve as a novel therapeutic target for MIRI.

Monitoring SARS-CoV-2 in sewage: toward sentinels with analytical accuracy

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effect of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested by electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.