One-step duplex RT-droplet digital PCR assay for the detection of norovirus GI and GII in lettuce and strawberry

诺如病毒 数字聚合酶链反应 生物 实时聚合酶链反应 复式(建筑) 检出限 重复性 逆转录聚合酶链式反应 分子生物学 聚合酶链反应 食品科学 病毒学 化学 色谱法 DNA 病毒 基因 遗传学 信使核糖核酸
作者
Chongzhen Sun,Jiayin Chen,Hui Li,Ling Fang,Shiwei Wu,Pallavi Jayavanth,Shuze Tang,Glòria Sánchez,Xiyang Wu
出处
期刊:Food Microbiology [Elsevier BV]
卷期号:94: 103653-103653 被引量:12
标识
DOI:10.1016/j.fm.2020.103653
摘要

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.
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