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. 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. Stool 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. Here 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.

中文翻译：

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粪便中 SARS-CoV-2 检测的优化方案

在 COVID-19 患者的粪便样本中检测到了 SARS-CoV-2，这对粪口传播具有潜在影响。与鼻咽拭子样本相比，粪便基质的复杂性给病毒检测带来了尚未解决的挑战。然而，需要稳健可靠的方法来估计 SARS-CoV-2 在肠道中的流行率和持久性，并确保基于微生物组的程序（例如粪便微生物群移植 (FMT)）的安全性。本研究的目的是建立一种灵敏可靠的方法来检测粪便样本中的 SARS-CoV-2。将不含 SARS-CoV-2 的个体的粪便样本在盐水缓冲液中匀浆，并加入已知滴度的灭活病毒，每 100 毫克粪便含有 50 至 750 个病毒颗粒。通过超滤浓缩病毒颗粒，提取 RNA，并使用 CDC 引物和探针通过实时逆转录聚合酶链反应 (RT-qPCR) 检测 SARS-CoV-2。我们使用的 RNA 提取程序可以通过 RT-qPCR 在大多数测试的粪便样本中检测到 SARS-CoV-2。每 100 毫克粪便中我们可以检测到少至 50 个病毒颗粒。然而，在低病毒滴度的样本中观察到了高变异性。针对 N1 区域的引物组提供了更可靠和精确的结果，对于该引物组，我们的方法的检测限为每毫克粪便 1 个病毒颗粒。在这里，我们描述了一种检测粪便样本中 SARS-CoV-2 的灵敏方法。该方法可用于确定粪便中SARS-CoV-2的持久性，并确保FMT等临床实践的安全性。