纳米磁分离实时荧光PCR定量检测登革2型病毒

    刘翌1，孙宁2，王飞1，田茵1，葛广路3，邓丛良1，张绍福1，陆琳1，汪琳1

    1.北京出入境检验检疫局，北京 100013；2.东南大学；3.国家纳米科学中心

    摘要：目的 建立一种登革2型病毒的快速提取和检测的方法。方法根据登革2型病毒的基因保守区，设计一套特异的实时荧光PCR的引物与探针，设计一对引物用于构建定量检测的标准品，以建立登革2型病毒实时荧光定量PCR检测方法；选用纳米磁微粒，建立纳米磁分离提取登革2型病毒RNA的方法，并对其核酸提取效果进行评估。结果纳米磁分离实时荧光PCR方法检测登革2型病毒具有快速、灵敏、特异的特点。在2.9×102~2.9×109copies/μl的范围内循环阀值（Ct）值与病毒拷贝数的对数值存在线性关系（R2 = 0.99）。结论建立的纳米磁分离实时荧光定量PCR方法能够快速准确地检测登革2型病毒，在口岸卫生检疫中具有很好的应用价值。

    关键词：磁性纳米微粒；实时荧光PCR；登革2型病毒

    中图分类号：R183.5　文献标识码：B

    Development of a new quantitative PCR assayof using magnetic

    nanoparticles separation for detection ofDengue virus 2 type

    LIU Yi*, SUN Ning, WANG Fei, TIAN Yin, GEGuang-lu, DENG Cong-liang,

    ZHANG Shao-fu, LU Lin, WANG Lin

    *Beijing Entry-Exit Inspection and QuarantineBureau, Beijing 100013, China

    Abstract:   Objective  To establish a real-time RT-qPCR assay of using magneticnanoparticles for rapid isolation and detection ofDV-2.   Methods   Sets of primers and probewere designed based on the conserved region of the E glycoproteinof DV-2 genome. The standard curve for quantification was set upfor DV-2 detection. The method based magnetic nanoparticles wasestablished to isolate nucleic acid of DV-2, and its effect ofnucleic acid separation was evaluated.  Results   The real-time PCR assay using magneticnanoparticles separation was showed high sensitivity andspecificity in detection of DV-2. The relationship betweenthreshold cycle (Ct) and logarithm of initial viral copies waslinear over a range of 2.9×102 to 2.9×109 copies/μl of DV-2 (R2 =0.99).  Conclusion   This method represents asensitive, specific, rapid approach to detect on DV-2 in clinicalspecimens.These results suggest that this technique may be usefulin early stage diagnosis of dengue infections at entry-exitfrontier ports.

    Key words:   Magneticnanoparticles; Real-time qPCR; DV-2

    《中国国境卫生检疫杂志》2013年6月刊