建立仙台病毒实时荧光重组酶等温扩增检测方法
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(1. 上海实验动物研究中心,上海 201203; 2. 上海海关,上海 200135)

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R-33

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Establishment of an isothermal real-time fluorescence recombinase polymerase amplification assay for detection of Sendai virus
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(1. Shanghai Laboratory Animal Research Center, Shanghai 201203, China.2. Shanghai Customs, Shanghai 200135)

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    摘要:

    目的 国标标准规定清洁级和SPF 级小鼠必须排除仙台病毒(Sendai virus, SeV)。为了提升效率,简化操作步骤,本研究建立了一种新型分子检测方法———实时荧光重组酶等温扩增技术(real-time RPA)用于检测SeV。方法 针对SeV 融合蛋白编码基因的保守序列,设计特异性扩增引物及探针并建立检测方法,并评价该检测方法的特异性、敏感性、稳定性和可靠性。最后将该检测方法和团标PCR 方法进行优势比较。结果 该方法具有很好的特异性,与小鼠肝炎病毒等六种病原微生物无交叉反应。其敏感性、稳定性和可靠性均可,并能够在25 min内完成,远远快于PCR 方法。结论 本研究建立的SeV 实时荧光RPA 是一种操作简单,高效直观且特异性好的检测方法。

    Abstract:

    Objective The Standardization of the People’s Republic of China stipulated that Sendai virus (SeV) must be excluded from clean and SPF grade mice. To simplify the detection and improve its efficiency, a novel real-time fluorescence recombinase polymerase amplification assay (real-time RPA) was developed for the detection of SeV in this study. Methods The specific primers and probes were designed against a conserved sequence of the gene encoding the SeV fusion protein, allowing the establishment of this detection method. The specificity, sensitivity, stability and reliability of the assay were evaluated. Finally, the superiority of this detection method was compared with the standard PCR method. Results The assay was highly specific, showing no cross-reactions with six other pathogenic microorganisms such as mouse hepatitis virus. The sensitivity, stability and reliability were acceptable, and this assay was completed within 25 minutes, much faster than the routinely used PCR. Conclusions In this study the SeV real-time RPA detection method is successfully established and found to be a simple, efficient, intuitive and specific detection method.

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冯丽萍,熊炜,高诚,魏晓锋.建立仙台病毒实时荧光重组酶等温扩增检测方法[J].中国比较医学杂志,2019,29(12):94~97.

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  • 收稿日期:2019-05-14
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  • 在线发布日期: 2020-01-13
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