纳米磁分离法结合实时荧光定量PCR检测恶性疟原虫的研究  

Detection of Plasmodium falciparum by using magnetic nanoparticles separation-based quantitative real-time PCR assay

作　　者：王飞[1] 田茵[1] 杨静[1] 孙福军[1] 孙宁[2] 刘必永 田睿[1] 葛广路[2] 邹明强[4] 邓丛良[1] 刘翌[1] 

机构地区：[1]北京国际旅行卫生保健中心,北京100094 [2]国家纳米科学中心 [3]江苏省盐城市疾病预防控制中心 [4]中国检验检疫科学研究院

出　　处：《中国血吸虫病防治杂志》2014年第5期522-525,530,共5页Chinese Journal of Schistosomiasis Control

基　　金：国家质检总局科研基金(2012IK218);国家重大科学研究计划(2011CB932803)

摘　　要：目的建立恶性疟原虫纳米磁分离实时荧光定量PCR检测方法,快速准确地检测恶性疟原虫,为输入性恶性疟防控提供技术支持。方法根据恶性疟原虫基因组18S rRNA保守区序列,设计并合成引物和探针;构建质粒标准品,拟合标准曲线,采用纳米磁分离法提取核酸,建立恶性疟原虫纳米磁分离实时荧光定量PCR检测法,并进行该方法的灵敏度和特异性评价。结果与常规法（镜检法和快检法）相比,该方法检测恶性疟原虫更加灵敏,并具有良好的特异性,在（2.5×101-2.9×108）copies/ml拷贝数范围内循环阈值（Ct值）与质粒标准品拷贝数的对数值之间存在良好的线性关系（R2=0.999）;应用该方法从非洲维和部队13名归国人员中检出1例低水平恶性疟原虫感染者,而常规法（镜检法和快检法）未检出。结论该方法能够快速准确地检测恶性疟原虫,在口岸低密度恶性疟原虫感染者的检测中具有很大的应用价值。Objective To establish a magnetic nanoparticles separation-based quantitative real-time PCR（RT-PCR）assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. Methods According to the conserved sequences of the P. falciparum genome 18SrRNA,the speciesspecific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard,fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. Results The relationship between the threshold cycle（Ct）and logarithm of initial templates copies was linear over a range of 2.5×101 to 2.5×108 copies/μl（R2=0.999）. Among 13 subjects of entry frontier,a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations（microscopic examinations and rapid tests）.Conclusion This assay shows a high sensitivity in detection of P. falciparum,with rapid and accurate characteristics,and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.

关 键 词：恶性疟原虫 输入性 纳米磁分离 实时荧光定量PCR 检测 

分 类 号：R382.31[医药卫生—医学寄生虫学]