Detection of GⅠ,GⅡNorovirus in Frozen Strawberries with Droplet Digital PCR

Objective:To establish a reverse transcription microdroplet digital PCR(RT-ddPCR)method for the detection of norovirus(GI and GII)in foods. Methods:Select primers according to ISO standards,optimize the reaction system,and anneal the temperature. Methodological experiments established a new method for rapid and simultaneous detection of GI and GII subtype norovirus in food. Results:The digital PCR was used to detect the annealing temperature of GI type norovirus at 56.5℃,and the annealing temperature of GII type norovirus was 58.1℃. The R²=0.9947 standard curve of GI plasmid standard was detected by RT-ddPCR,and the R²=0.9950 of the standard curve of GII plasmid standard by RT-ddPCR showed that the method had a good linear relationship. Compared with the RT-qPCR sensitivity,the sensitivity of the RT-ddPCR method was an order of magnitude higher than that of the RT-qPCR method. Within the detection range,the minimum detection limit was as low as the unit of copies. The minimum RSD was 3.8%,indicating that the experiment was reproducible. Conclusion:The norovirus digital PCR method established in this study had the advantages of high specificity,high sensitivity and low detection limit. It could be used for the quantitative detection of norovirus in frozen strawberries.