Abstract
Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.
Highlights
Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared
While many vendors provide synthetic SARSCoV-2 RNA featuring gene targets recommended by the United States Centers for Disease Control and Prevention (CDC)[17] and the German Centre for Infection Research (DZIF)[18], preliminary studies have revealed that not all of them are at reliable concentrations[19]
RNA-dependent RNA polymerase protein (RdRP) proves to be a poor target for the American Type Culture Collection (ATCC) synthetic SARS-CoV-2 RNA under given reaction conditions, since detection is decreased by an order of magnitude in the droplet digital PCR (ddPCR) assay and the RT-qPCR reaction efficiency is compromised (114%)
Summary
Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. While the primer/probe sets used are generally consistent, classifying samples as positive for the presence of SARS-CoV-2 RNA has often been based on arbitrary thresholds set in the absence of a relevant standard curve[14,15,16] These experimental inconsistencies and the lack of a clearly validated experimental pipeline contribute significantly to heterogeneity in detection and quantification of viral RNA in stool. To overcome these challenges, we sought to test a variety of accessible and common methods for the preservation, extraction, and detection of viral RNA from stool samples, and present here an optimized pipeline. Our findings here will guide the field toward a more standardized method of robustly measuring the fecal burden of SARS-CoV-2 RNA both in clinical and research settings
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