Department of Parasitology, Medical College of Soochow University, Suzhou 215123, People's Republic of China.
Parasit Vectors. 2011 Jun 21;4:115. doi: 10.1186/1756-3305-4-115.
Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions.
A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection.
The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method.
This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.
环介导等温扩增（LAMP）是一种高灵敏度的 DNA 检测方法，有望用于检测包括疟原虫在内的传染病病原体的分子检测。然而，在大多数疟疾流行地区，由于难以准确解释结果，存在扩增产物敏感可视化的问题，以及由于扩增 DNA 数量高而导致污染的风险，当前的 LAMP 方法在诊断上不可行。在这项研究中，我们建立了一种新型的闭管系统可视化 LAMP 方法，并在模拟现场条件下验证了其对疟疾的诊断。
在正常 LAMP 反应前加入含有高灵敏度 DNA 荧光染料 SYBR Green I 的微晶蜡染料胶囊，建立可视化 LAMP 方法。总共采集了 89 份滤纸血样，采用简单煮沸法提取 DNA，并采用可视化 LAMP 方法检测滤纸血样中是否存在间日疟原虫感染。
蜡胶囊在等温扩增过程中保持完整，只有在扩增后温度升高到熔点时才会将 DNA 染料释放到反应混合物中。在冷却后不久，凝固的蜡将反应混合物密封在管底，从而最大限度地降低了气溶胶污染的风险。与显微镜检查相比，LAMP 的灵敏度和特异性分别为 98.3%（95%置信区间：91.1-99.7%）和 100%（95%置信区间：88.3-100%），与巢式聚合酶链反应方法高度一致。
这种新型、廉价、快速的可视化 LAMP 方法在资源有限的现场环境中进行疟疾诊断是可行的。
