Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

Abstract

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

Standardization of laboratory practices for the study of the human gut microbiome

“…To prevent this, several methods including different stabilization buffers have been developed in the recent years [38,40,41]. In line with the results reported by Roesch et al [42], we found a higher amount of Enterobacteriaceae in samples stored at RT for 3 -5 days, when compared to the same sample when directly frozen.…”

“…Collecting and transporting samples at room temperature however promotes the overgrowth of facultative anaerobes and potential degradation of nucleic acids by breakdown of strict anaerobes. To prevent this, several methods including different stabilization buffers have been developed in the recent years [38, 40, 41]. In line with the results reported by Roesch et al [42], we found a higher amount of Enterobacteriaceae in samples stored at RT for 3 – 5 days, when compared to the same sample when directly frozen.…”

Using haematophagous fly blood meals to study the diversity of blood‐borne pathogens infecting wild mammals

“…Finally, noninvasive sampling of faecal droppings, for example, can be very difficult to collect fresh enough from cryptic and/or evasive animals and such specimens do not necessarily allow satisfactory detection of all pathogens. Furthermore, these samples often represent difficult media for pathogen detection due to nucleic acid or protein degradation and also because they contain inhibitors of the extraction or amplification reagents required for molecular analysis (Natarajan et al, 2021 ; Sarabia et al, 2020 ). New complementary approaches are therefore needed for monitoring and detecting pathogens in wildlife.…”