Abstract: Objective: To develop a quantitative detection method for norovirus (NoV) in foods based on digital PCR. Methods: Three sets of specific primers and probes were selected to establish one-step reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR) assays for NoV genotype I (GI), genotype II (GII) and MS2 phage, respectively. The specificity of these RT-ddPCR assays was verified. Their limit of quantitation, accuracy and repeatability were analyzed using NoV RNA reference materials and MS2 phage RNA. Artificially positive samples were prepared to analyze the influence of MS2 phage on the results of NoV detection. Results: The three RT-ddPCR assays had satisfactory specificity, accuracy and repeatability. The limit of quantitation of NoV RT-ddPCR was at five orders of magnitude from 104 to 100 copies/μL and close to that of MS2 phage RT-ddPCR. There was no significant difference between the quantitative results for artificially positive samples with and without added MS2 phage (P ≥ 0.05). Conclusion: NoV RNA could be accurately quantified by the NoV RT-ddPCR assay. MS2 phage did not affect the quantitative results of NoV, and could be added as a process control model virus to indicate virus recovery and calculate the content of NoV particles in foods.

Key words: reverse transcriptase droplet digital polymerase chain reaction; norovirus; MS2 phage; recovery