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

Aravind Natarajan1,2, Alvin Han3, Soumaya Zlitni1,2, Erin F Brooks2, Summer E Vance2, Marlene Wolfe4, Upinder Singh5, Prasanna Jagannathan3,5, Benjamin A Pinsky5,6, Alexandria Boehm4, Ami S Bhatt7,8

  1. Department of Genetics, Stanford University, Stanford, CA, USA.
  2. Department of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, CA, USA.
  3. Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA.
  4. Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA.
  5. Department of Medicine (Infectious Diseases and Geographic Medicine), Stanford University, Stanford, CA, USA.
  6. Department of Pathology, Stanford University, Stanford, CA, USA.
  7. Department of Genetics, Stanford University, Stanford, CA, USA. asbhatt@stanford.edu.
  8. Department of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, CA, USA. asbhatt@stanford.edu.

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.

Presented By Aravind Natarajan and Alvin Han

COVID-19, MedicineNicholas Liao2021-10 (Oct)