An optimised protocol for detection of SARS-CoV-2 in stool

Tianqi Li,Jacob Scadden,Lizbeth Sayavedra,Daniel A Yara,Enriqueta Garcia-Gutierrez,Arjan Narbad,Chloe Hutchins,Alp Aydin,Justin O’Grady,Jade Davies,Stefano Romano

doi:10.1186/s12866-021-02297-w

Abstract

BackgroundSARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples.ResultsStool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool.ConclusionsHere we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.

Highlights

SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission
Several recent studies indicate that viable SARS-CoV-2 can be detected in stool samples of COVID-19 patients [6, 7], suggesting that a possible risk for faecal-oral transmission exists
We describe a robust approach to detect SARSCoV-2 in stool samples, having a limit of detection (LoD) of 1 and 2 vp mg− 1 for the N1 and N2 primer set, respectively

Summary

Introduction

SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. Robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples. Robust and reliable methods are an urgent need, as microbiota-based therapies such as faecal microbiota transplantation (FMT) would need to rely heavily on the accurate screening of donor stools to ensure the absence of SARS-CoV-2 and guarantee patient safety [16]

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