A broadly reactive one-step SYBR Green I real-time RT-PCR assay for rapid detection of murine norovirus.
A broadly reactive one-step SYBR Green I real-time RT-PCR assay for rapid detection of murine norovirus.
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DOI：
10.1371/journal.pone.0098108
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Kyuwa S
中科院分区：
文献类型：
--
作者：
Hanaki K;Ike F;Kajita A;Yasuno W;Yanagiba M;Goto M;Sakai K;Ami Y;Kyuwa S
建立了一种一步法SYBR Green I实时RT-PCR方法，用于检测和定量检测多种小鼠诺如病毒(MNV)。根据MNV1−ORF2结合区10 1个序列的多重序列比对，设计了MNVORF2的多重序列比对方法。用7个MNV质粒验证了该方法的广谱反应性和定量能力。检测在1h内完成，可靠的检测下限为10个拷贝的MNV质粒或每毫升RAW264细胞培养扩增病毒的组织培养感染量的中位数为0.063。使用158份小鼠粪便样本对该方法的诊断性能进行了评估，其中91份被确认为阳性。熔融曲线分析证实了样品中MNV的多样性。这是首次报道一种广泛反应的一步法SYBR Green I实时RT-PCR检测MNV。这种快速而灵敏的检测方法使其成为诊断应用的有力工具。
A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.
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