The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/μL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/μL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 10⁶ to 1.70 × 10⁸ copies and 4.80 × 10⁵ to 2.50 × 10⁷ copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 10⁴ copies/25g (NoV GI) and 2.36 × 10⁴ ciopies/25g (NoV GII), and that in strawberry was 2.56 × 10⁴ copies/25g (NoV GI) and 2.64 × 10⁴ ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV.

该研究旨在开发一种敏感的一步双工逆转录液滴数字聚合酶链反应（RT-ddPCR），以检测莴苣和草莓中的诺如病毒基因组I和II（NoV GI和GII）。将测定的特异性，灵敏度，可重复性和鲁棒性与RT-qPCR进行了比较。RT-ddPCR检测到的NoV GI和NoV GII的最低浓度分别为4.68和8.47拷贝/μL，远低于RT-qPCR的最低浓度，分别为46.8和84.7拷贝/μL。生菜和草莓样品被NoV GI和GII悬浮液人工污染，接种量为3.00×10 ⁶至1.70×10 ⁸份和4.80×10 ⁵至2.50×10 ⁷分别。低接种量的草莓掺入物通过RT-ddPCR显示阳性结果，而通过RT-qPCR显示阴性。同时，RT-ddPCR还显示出生菜和草莓中NoV的平均回收率高于RT-qPCR.RT-ddPCR对生菜中NoV的检测限（LoDs）为2.32×10 ⁴拷贝/ 25g（NoV GI）和25g（NoV GII）分别为2.36×10 ⁴ ciopies和25g（NoV GI）的草莓为2.56×10 ⁴拷贝/ 25g（NoV GI）和2.64×10 ⁴ciopies / 25g（NoV GII），比RT-qPCR低10倍。已开发的双工RT-ddPCR检测方法具有稳定性，并提高了低NoV浓度的食品样品中抗抑制剂的能力，因此与RT-qPCR相比，它是避免假阴性结果的可靠方法。总之，本研究开发的一步式RT-ddPCR方法与检测食源性病毒（如NoV）有关。