SARS-CoV-2 surface contamination in staff common areas and impact on healthcare worker infection: prospective surveillance during the COVID-19 pandemic

We prospectively surveyed SARS-CoV-2 RNA contamination in staff common areas within an acute-care hospital. An increasing prevalence of surface contamination was detected over time. Adjusting for patient census or community incidence of coronavirus disease 2019 (COVID-19), the proportion of contaminated surfaces did not predict staff COVID-19 infection on study units.

SARS-CoV-2 RNA and Supermarket Surfaces: A Real or Presumed Threat?

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in March 2020 in Italy, leading to the pandemic of coronavirus disease 2019 (COVID-19) that continues to cause high global morbidity and mortality in human populations. Numerous studies have focused on the spread and persistence of the virus in the hospital setting. New scientific evidence shows that SARS-CoV-2 is present in different community environments. Although aerosol is one of the main routes of transmission for SARS-CoV-2, indirect contact through virus-contaminated surfaces could also play a key role. The survival and persistence of SARS-CoV-2 on surfaces appear to be influenced by the characteristics of the material, temperature, and humidity. In this study, we investigated the presence of SARS-CoV-2 RNA on surfaces in 20 supermarkets throughout the Apulia region during the lockdown period. We collected a total of 300 swab samples from various surfaces including supermarket scales, trolley handles, refrigerator and freezer handles, and keyboards. In total, 13 (4.3%) surfaces were positive for SARS-CoV-2 RNA contamination, with shopping trolley handles being the most frequently contaminated. This study showed that contamination in public spaces can occur, so we remark the importance to adopt adequate preventive measures, including environment ventilation, careful surfaces sanitation, hand hygiene, and correct usage of masks, to reduce the likelihood of virus transmission.

The Problem of DNA/RNA Contamination in the Laboratory during PCR Testing for COVID-19

Introduction. When conducting PCR (polymerase chain reaction) testing of biospecimens for SARS-CoV-2 RNA at the beginning of the COVID-19 pandemic, the laboratory service in Russia and foreign countries encountered problems related to the accuracy of diagnostics and obtaining false negative, false positive, and dubious results. The objective of this work was to analyze current literature on the problem of false positive and dubious results of RT-PCR testing for COVID-19. Material and methods. We selected Russian and foreign English-language publications devoted to organization of laboratory diagnostics of the novel coronavirus disease, challenges of PCR testing for SARS and MERS, and general issues of DNA contamination in a PCR laboratory for 2012–2020. We also reviewed current regulations and guidelines for COVID-19 diagnostic testing. Results. The analysis of factors leading to contamination of specimens with nucleic acids in the laboratories performing massive COVID-19 PCR testing during the pandemic showed that the main reasons for contamination included a large number of tests, accumulation of samples in the laboratory, and the increased amount of wastes containing amplification products. Cross-contamination occurs due to technical errors in the course of laboratory manipulations at the stages of sample preparation and inactivation, RNA isolation, and addition of cDNA/RNA or positive control samples to the reaction mixture. Pollution of laboratory working areas with amplicons arising from the opening of tubes and plates containing PCR products is the main cause of total contamination in the laboratory. Signs of cross-contamination include the increase in the proportion of positive samples with low threshold cycle values and detection of a positive signal from negative control samples at RNA isolation and amplification stages. A positive result for all samples in a round, including negative control samples, is a marker of “total contamination” in the laboratory. In addition to contamination, formation of nonspecific PCR products at late reaction cycles and nonspecific fluorescence of the reaction mixture, which occurs when reagent storage temperatures are not observed, may also lead to false positive results. Conclusion. To prevent contamination in a PCR laboratory, strict control over the flow of test samples and medical wastes, regular analysis of the frequency of positive test results, and mandatory laboratory quality control of testing and DNA/RNA contamination are compulsory.

High-purity DNA extraction from animal tissue using picking in the TRIzol-based method

TRIzol is used for the extraction of RNA, DNA and proteins from tissues or cells. Here, we present a simple picking method to extract DNA from tissues using TRIzol. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the picking method's DNA is ideal and better than that obtained by the classic TRIzol method. Gel electrophoresis showed that there was no RNA contamination, and the DNA had not degraded. DNA extracted by the picking method had the same performance in restriction enzyme digestion and quantitative PCR as that obtained by the traditional method. Viral DNA in the infected tissue was also obtained. This modified method facilitates various molecular biology assays.

SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data

Background Droplet-based single-cell RNA sequence analyses assume that all acquired RNAs are endogenous to cells. However, any cell-free RNAs contained within the input solution are also captured by these assays. This sequencing of cell-free RNA constitutes a background contamination that confounds the biological interpretation of single-cell transcriptomic data. Results We demonstrate that contamination from this "soup" of cell-free RNAs is ubiquitous, with experiment-specific variations in composition and magnitude. We present a method, SoupX, for quantifying the extent of the contamination and estimating "background-corrected" cell expression profiles that seamlessly integrate with existing downstream analysis tools. Applying this method to several datasets using multiple droplet sequencing technologies, we demonstrate that its application improves biological interpretation of otherwise misleading data, as well as improving quality control metrics. Conclusions We present SoupX, a tool for removing ambient RNA contamination from droplet-based single-cell RNA sequencing experiments. This tool has broad applicability, and its application can improve the biological utility of existing and future datasets.

No evidence for DNA N6-methyladenine in mammals

N6-methyladenine (6mdA) is a widespread DNA modification in bacteria. More recently, 6mdA has also been characterized in mammalian DNA. However, measurements of 6mdA abundance and profiles are often very dissimilar between studies, even when performed on DNA from identical mammalian cell types. Using comprehensive bioinformatics analyses of published data and novel experimental approaches, we reveal that efforts to assay 6mdA in mammals have been severely compromised by bacterial contamination, RNA contamination, technological limitations, and antibody nonspecificity. These complications render 6mdA an exceptionally problematic DNA modification to study and have resulted in erroneous detection of 6mdA in several mammalian systems. Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals.

SoupX removes ambient RNA contamination from droplet based single-cell RNA sequencing data

BackgroundDroplet based single-cell RNA sequence analyses assume all acquired RNAs are endogenous to cells. However, any cell free RNAs contained within the input solution are also captured by these assays. This sequencing of cell free RNA constitutes a background contamination that confounds the biological interpretation of single-cell transcriptomic data.ResultsWe demonstrate that contamination from this ‘soup’ of cell free RNAs is ubiquitous, with experiment-specific variations in composition and magnitude. We present a method, SoupX, for quantifying the extent of the contamination and estimating ‘background corrected’ cell expression profiles that seamlessly integrate with existing downstream analysis tools. Applying this method to several datasets using multiple droplet sequencing technologies, we demonstrate that its application improves biological interpretation of otherwise misleading data, as well as improving quality control metrics.ConclusionsWe present ‘SoupX’, a tool for removing ambient RNA contamination from droplet based single cell RNA sequencing experiments. This tool has broad applicability and its application can improve the biological utility of existing and future data sets.Key PointsThe signal from droplet based single cell RNA sequencing is ubiquitously contaminated by capture of ambient mRNA.SoupX is a method to quantify the abundance of these ambient mRNAs and remove them.Correcting for ambient mRNA contamination improves biological interpretation.