Department of Food Science and Engineering, Jinan University, 601 Huangpu Avenue, Guangzhou, 510632, China.
Institute of Pathogenic Microbiology, Guangdong Provincial Center for Disease Control and Prevention, 160 Qunxian Road, Guangzhou, 511430, China.
Food Microbiol. 2021 Apr;94:103653. doi: 10.1016/j.fm.2020.103653. Epub 2020 Oct 6.
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 × 10 to 1.70 × 10 copies and 4.80 × 10 to 2.50 × 10 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 × 10 copies/25g (NoV GI) and 2.36 × 10 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 10 copies/25g (NoV GI) and 2.64 × 10 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.
本研究旨在开发一种灵敏的一步法双重逆转录液滴数字聚合酶链反应（RT-ddPCR），用于检测生菜和草莓中的诺如病毒基因组 I 和 II（NoV GI 和 GII）。该方法的特异性、灵敏度、重复性和稳健性与 RT-qPCR 进行了比较。RT-ddPCR 检测 NoV GI 和 NoV GII 的最低浓度分别为 4.68 和 8.47 拷贝/μL，明显低于 RT-qPCR 的 46.8 和 84.7 拷贝/μL。生菜和草莓样品被人工污染有 NoV GI 和 GII 悬液，接种量分别为 3.00×10 至 1.70×10 拷贝和 4.80×10 至 2.50×10 拷贝。用 RT-ddPCR 检测低接种量的草莓样品呈阳性，而用 RT-qPCR 检测结果为阴性。同时，RT-ddPCR 检测生菜和草莓中诺如病毒的平均回收率也高于 RT-qPCR。RT-ddPCR 检测生菜中 NoV 的检测限（LoD）为 2.32×10 拷贝/25g（NoV GI）和 2.36×10 拷贝/25g（NoV GII），草莓中的检测限为 2.56×10 拷贝/25g（NoV GI）和 2.64×10 拷贝/25g（NoV GII），均比 RT-qPCR 低 10 倍。与 RT-qPCR 相比，开发的双重 RT-ddPCR 检测方法在低浓度诺如病毒的食品样本中表现出稳定性和更高的抗抑制能力，从而避免了假阴性结果，是一种可靠的方法。总之，本研究建立的一步法 RT-ddPCR 方法适用于检测食源性病原体，如诺如病毒。
