Abstract: Objective: To develop a rapid and sensitive one-step reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay for the detection of norovirus genogroup II (NoV GII) in lettuce. Methods: The RT-ddPCR system was optimized; the detection limit was tested using a series of 10-fold diluted enzyme digested plasmids containing the target gene sequence of NoV GII. The specificity was validated by using nucleic acids from Nov GII and other enteric viruses as targets. The repeatability of the method was also examined. Lettuce samples artificially infected with different concentrations (high, moderate, and low levels) of NoV GII were detected by RT-ddPCR following RNA extraction performed as described in ISO/TS 15216-1:2013. The influence of removal of inhibitors on the performance of RT-ddPCR was evaluated in comparison with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: The maximum and minimum detection limits of RT-ddPCR were 8.47 × 104 and 2.12 copies/μL, respectively. The amplification efficiency was 95.44% (r = 0.997 3). The existence of inhibitors in lettuce showed no significant difference in the recovery RT-ddPCR at different contamination levels. The recovery rates of RT-qPCR at low contamination level with and without inhibitors were 1.43% and 9.71% respectively, which were significantly different from RT-ddPCR (11.80% and 12.53% respectively). Conclusions: The RT-ddPCR assay developed was stable in detecting food samples regardless of the presence of inhibitors such as lettuce. It could efficiently detect low concentrations of NoV GII virus in food without false-negative results, which may occur in RT-qPCR because of the existence of inhibitors. Therefore, this one-step RT-ddPCR assay was able to detect low levels of NoV GII in contaminated food samples, which indicates a considerable application prospect in the field of foodborne virus detection.

Key words: reverse transcription droplet digital PCR (RT-ddPCR), one-step procedure, norovirus genogroup II (NoV GII), lettuce, recovery rate