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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 establish a sensitive and sensitive method for the detection of Plasmodium falciparum based on Loop-mediated Isothermal Amplification (LAMP). Methods: The LAMP primer was designed according to the conserved region of ribosomal DNA of Plasmodium falciparum, and the ring-mediated isothermal amplification method was established by optimizing the Mg2 +, betaine concentration and reaction temperature in LAMP system. Products were detected, the test results can be directly observed by visual SYBR Green I fluorescent color determination. Results: The method can detect 70 copies / tube of Plasmodium falciparum nucleic acid fragments, and has high specificity, can distinguish between detection of common blood virus. The method has the following advantages: 1, the entire reaction is carried out at constant temperature, without the need of a thermal cycler; 2, the closed-tube test greatly reduces the risk of cross-contamination of the amplified product; 3, the test speed is fast; Conclusion: The establishment of this method provides a simple, sensitive and highly specific tool for quick screening of Plasmodium falciparum in the field.