单管可视化环介导等温扩增技术快速检测恶性疟原虫  

A Closed-tube Isothermal Amplification Method for Highly Sensitive and Visualized Detection of Plasmodium Falciparum

作　　者：江再茂[1] 马雪萍[2,3] 殷竹君[4] 陆妍[2,3] 曹晓梅[3] 武海萍[2] 

机构地区：[1]中国人民解放军第171医院,江西九江332000 [2]南京军区军事医学研究所,江苏南京210002 [3]南京军区南京总医院药理科,江苏南京210002 [4]江苏省出入境检验检疫局,江苏南京210001

出　　处：《现代生物医学进展》2014年第26期5014-5018,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81373485);江苏省科技支撑计划-社会发展项目(BE2011607)

摘　　要：目的:建立一种基于环介导等温核酸扩增技术(Loop-mediated Isothermal Amplification,LAMP)的恶性疟原虫高灵敏可视化闭管检测方法。方法:针对恶性疟原虫核糖体DNA的序列保守区设计LAMP引物,通过优化LAMP体系中的Mg2+、甜菜碱浓度和反应温度等因素,建立环介导等温扩增法;并结合蜡封反应管对产物进行检测,检测结果可直接通过肉眼观察SYBR Green I荧光显色进行判定。结果:本方法可检测到70个拷贝/管的恶性疟原虫核酸片段,并具有高特异性,可区分检测常见的血液病毒。该法具有如下优点:1、整个反应恒温进行,无需热循环仪;2、闭管检测,极大降低了扩增产物交叉污染的风险;3、检测速度快,整个检测过程只需30 min。结论:该法的建立为恶性疟原虫的现场快速筛检提供了一种简便、高灵敏、高特异的工具。Objective： To develop a visualized closed-tube loop-mediated isothermal amplification （LAMP） assay with a high sensitivity for the detection of Plasmodium falciparum. Methods： LAMP primers were desighed based on the conserved region of the ribosomal DNA of Plasmodium falcipamm. LAMP reaction conditions were optimized in light of the concentration of Mg^2＋ and betaine, as well as the reaction temperature. The read-out was carried out by using naked eyes to observe the color changes of fluorescent intercalator SYBR Green I, which was sealed by wax at the bottom of the detection tube before LAMP reaction. Results： The sensitivity of the proposed method is high and can test as low as 70 copies of genomic DNA of Plasmodium falciparum. The method is highly specific, and can easily distinguish Plasmodium falciparum from common blood pathogens. This method has the following advantages： 1. LAMP assay is an isothermal amplification,so no thermal cycler is needed; 2. The closed-tube LAMP reaction greatly decrease the risk of cross-contamination from amplicons; 3. The assay is very fast, and only 30 minutes is needed for the whole amplification process. Conclusions： The proposed method is a simple, highly sensitive and highly sepefic tool for rapid screening of Plasmodium falciparum.

关 键 词：恶性疟原虫 环介导等温扩增 闭管反应 

分 类 号：R-446[医药卫生] R446.11