Detection of Foodborne RNA Viruses by Reverse Transcriptase Droplet Digital PCR

Foodborne viruses remain the largest cause of human gastroenteritis and one of the largest contributors to foodborne illnesses worldwide. Currently, quantitative reverse transcription PCR (qRT-PCR) or real-time qPCR are the detection methods commonly used for quantification of foodborne viruses, but those methods have several disadvantages, such as relying on standard curves for quantification and the background noise from a bulk reaction. ddPCR uses an oil-water emulsion to form multiple droplets that partition small amounts of viral genetic material (DNA or RNA) into each of the droplets. These droplets then undergo amplification cycles and are analyzed using Poisson distributions. This allows for absolute quantification without the need for a standard curve, which makes ddPCR a precise tool in surveillance of foodborne viruses. Herein, we describe the process of detecting foodborne viruses using RNA isolated from various matrices. Up to 96 samples including the positive and negative controls can be analyzed on a single plate by ddPCR.