纳米磁分离实时荧光PCR定量检测登革2型病毒  

Development of a new quantitative PCR assay of using magnetic nanoparticles separation for detection of Dengue virus 2 type

作　　者：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] 

机构地区：[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

出　　处：《中国国境卫生检疫杂志》2013年第3期145-149,172,共6页Chinese Journal of Frontier Health and Quarantine

基　　金：国家重大科学研究计划项目(2011CB932803);国家质检总局科研基金项目(2010IK210)

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（C）t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。Objective To establish a real-time RT-qPCR assay of using magnetic nanoparticles for rapid isolation and detection of DV-2. Methods Sets of primers and probe were designed based on the conserved region of the E glycoprotein of DV-2 genome. The standard curve for quantification was set up for DV-2 detection. The method based magnetic nanoparticles was established to isolate nucleic acid of DV-2, and its effect of nucleic acid separation was evaluated. Results The real-time PCR assay using magnetic nanoparticles separation was showed high sensitivity and specificity in detection of DV-2. The relationship between threshold cycle （C,） and logarithm of initial viral copies was linear over a range of 2.9×10^2 to 2.9×10^9 copies/μl of DV-2 （R2= 0.99）. Conclusion This method represents a sensitive, specific, rapid approach to detect on DV-2 in clinical specimens.These results suggest that this technique may be useful in early stage diagnosis of dengue infections at entry-exit frontier ports.

关 键 词：磁性纳米微粒 实时荧光PCR 登革2型病毒 

分 类 号：R183.5[医药卫生—流行病学]