Extraction of Human Coronavirus 229E from Feces for RT-qPCR of the Nucleocapsid Gene Using the Bead Ruptor Elite

Human coronavirus 229E (HCoV-229E) is an alpha-coronavirus transmitted primarily via respiratory droplets and fomites [1]. However, this virus - as well as many other coronaviruses - has been detected in fecal samples of asymptomatic patients, indicating a potential for fecal-oral transmission after symptomatic infection subsides [2]. Additionally, some coronaviruses appear in higher viral titers in feces than they do in respiratory samples, which could prove to be a great source of viral analytes for extraction. As an enveloped, positive sense, single stranded RNA virus primarily infecting humans and bats, this virus is a strong model system for studying the novel SARS-CoV-2 in a BSL-2 setting [1]. Herein, we have used HCoV-229E to model RNA extraction procedures from fecal samples that have been spiked to clinically-relevant concentrations [3], demonstrating how the Omni Bead Ruport Elite bead mill homogenizer can be implemented into an extraction and RT-qPCR workflow for research studies investigating coronavirus infections.

Table 2. Quantification of RT-qPCR results for N gene amplification from HCoV-229E spiked fecal samples. Results coordinate with the amplification visualization displayed in Figure 1. 

1. Fecal sample extractions after being spiked with 2.0 x 106 PFU/mL of virus. 
2. Fecal sample extractions after being spiked with 2.0 x 105 PFU/mL of virus. 
3. Fecal sample extractions after being spiked with 2.0 x 104 PFU/mL of virus. 
4. Negative controls for fecal samples run through the extraction process without any viral spike.