Evaluating Viral Process Controls: Turnip Crinkle Virus and Tulane Virus Demonstrate Similar RNA Extraction Efficiencies to Norovirus Genogroups I and II

Introduction: Noroviruses are a leading cause of foodborne disease. Historically, noroviruses have been difficult to detect in food and environmental samples, in part due to low concentrations and the lack of a cell culture system. RT-qPCR is a powerful tool for detection of noroviruses; however, processing complex samples in preparation for RT-qPCR often results in low and variable virus recovery efficiencies. 

Purpose: This study evaluated several viruses for their utility as process (extraction) controls for human noroviruses during the steps of RNA extraction and RT-qPCR.

Methods: Several viruses, including murine norovirus (MNV), Tulane virus (TV), turnip crinkle virus (TCV), and an F-RNA coliphage (MS2), were compared to a GI.6 and a GII.4 human norovirus relative to recovery efficiency using the BioMérieux Nuclisens easyMAG RNA extraction system and an Invitrogen SuperScript III Platinum One-Step qRT-PCR kit. All viruses were compared at a range of input virus from 2 – 7 log genome equivalent copies (GEC) per 100 µl of DEPC-treated water.

Results: NoV GI.6 and GII.4 had similarly high recoveries (losses of -0.2 ± 0.4 and 0.0 ± 0.3 log GEC, respectively) at all input levels. The virus with the most similar recovery efficiency was TCV, which had losses of 0.0 ± 0.6 log GEC at lower viral inputs (P > 0.05) and losses of 0.7 ± 0.7 log GEC at higher inputs. MNV had the lowest recovery efficiency, with losses of 1.5 ± 0.6 log GEC across all inputs.

Significance: With similar recovery efficiencies to human norovirus GI.6 and GII.4, TCV and TV may serve as appropriate process controls for extracting and detecting noroviruses from food and environmental samples. Use of validated process controls would increase confidence in detection results and facilitate quantification of virus load, both of which are important to food safety monitoring and assessment.