Monteiro Silvia, Santos Ricardo
Instituto Superior Técnico, Laboratório Analises, Universidade Lisboa, Lisbon, Portugal.
PLoS One. 2017 Jul 27;12(7):e0179985. doi: 10.1371/journal.pone.0179985. eCollection 2017.
Sensitive detection of water- and foodborne enteric viruses is extremely relevant, especially due to the low concentrations in which they are found. Accurate and sensitive detection of Norovirus, the primary responsible for water- and foodborne outbreaks, is of particular importance. Quantification of Norovirus is commonly performed by quantitative RT-PCR (RT-qPCR). In recent years a new platform was developed, digital PCR, that quantifies without the need for a standard curve thus decreasing the errors associated with its utilization. The platform developed by LifeTechnologies, QuantStudio 3D Digital PCR is amongst the least studied digital platform and although it allows the direct detection of DNA targets it requires a two-step RT-PCR for the detection of RNA targets. In this work we developed a new protocol able to detect Norovirus using a one-step digital PCR reaction (RT-dPCR). The performance of the newly developed one-step digital PCR was compared to RT-qPCR for the detection of Norovirus genogroup I and genogroup II. The sensitivity of RT-dPCR was identical to that of RT-qPCR, and the quantitative data determined by both methods were not significantly different for most samples. This one-step absolute quantification approach is a useful tool to minimize the time spent currently using this particular platform to amplify viral RNA and to standardize quantification of enteric viruses in food and environmental samples. This study proved the usefulness of the newly developed RT-dPCR protocol for a sensitive and accurate detection of low-copy targets.
对水传播和食源性肠道病毒进行灵敏检测极为重要，尤其是考虑到它们在极低浓度下被发现的情况。准确灵敏地检测诺如病毒（水传播和食源性疫情的主要病原体）尤为重要。诺如病毒的定量通常通过定量逆转录聚合酶链反应（RT-qPCR）进行。近年来，一种新的平台——数字PCR被开发出来，它无需标准曲线即可进行定量，从而减少了使用过程中产生的误差。LifeTechnologies公司开发的QuantStudio 3D数字PCR平台是研究最少的数字平台之一，尽管它能够直接检测DNA靶点，但检测RNA靶点时需要两步逆转录聚合酶链反应。在这项工作中，我们开发了一种新方案，能够通过一步数字PCR反应（RT-dPCR）检测诺如病毒。将新开发的一步数字PCR的性能与RT-qPCR用于检测诺如病毒基因I组和基因II组的性能进行了比较。RT-dPCR的灵敏度与RT-qPCR相同，两种方法测定的定量数据对大多数样品来说没有显著差异。这种一步绝对定量方法是一种有用的工具，可最大限度地减少目前使用该特定平台扩增病毒RNA所花费的时间，并使食品和环境样品中肠道病毒的定量标准化。这项研究证明了新开发的RT-dPCR方案对于灵敏准确地检测低拷贝靶点的有用性。
